Hiv-1 env fusion peptide nanoparticle carrier conjugates and their use

ABSTRACT

Embodiments of immunogenic conjugates including the HIV-1 Env fusion peptide and methods of their use and production are disclosed. In several embodiments, the immunogenic conjugates can be used to generate an immune response to HIV-1 Env in a subject, for example, to treat or prevent an HIV-1 infection in the subject.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/735,188, filed Sep. 23, 2018, which is incorporated by reference inits entirety.

FIELD

This disclosure relates to immunogenic conjugates including HIV-1envelope (Env) fusion peptides conjugated to a self-assembling proteinnanoparticle carrier and their use to induce an immune response in asubject.

BACKGROUND

Millions of people are infected with HIV-1 worldwide, and 2.5 to 3million new infections have been estimated to occur yearly. Althougheffective antiretroviral therapies are available, millions succumb toAIDS every year, especially in sub-Saharan Africa, underscoring the needto develop measures to prevent the spread of this disease.

An enveloped virus, HIV-1 hides from humoral recognition behind a widearray of protective mechanisms. The major envelope protein of HIV-1 is aglycoprotein of approximately 160 kD (gp160). During infection,proteases of the host cell cleave gp160 into gp120 and gp41. Gp41 is anintegral membrane protein, while gp120 protrudes from the mature virus.Together gp120 and gp41 make up the HIV-1 Env spike, which is a targetfor neutralizing antibodies.

It is believed that immunization with an effective immunogen includingepitopes of the HIV-1 Env glycoprotein can elicit a neutralizingresponse, which may be protective against HIV-1 infection. However,despite extensive effort, a need remains for agents capable of suchaction.

SUMMARY

This disclosure provides novel immunogenic conjugates for eliciting animmune response to HIV-1 Env in a subject.

The immunogenic conjugates comprise a self-assemblingprotein-nanoparticle carrier conjugated to HIV-1 Env fusion peptides.The self-assembling protein-nanoparticle carrier is comprised of amultimer of fusion proteins. Each fusion protein in the multimercomprises a self-assembling protein nanoparticle subunit fused to aheterologous carrier protein. The fusion proteins self-assemble to formthe self-assembling protein-nanoparticle carrier. The HIV-1 Env fusionpeptides conjugated to the self-assembling protein-nanoparticle carrier,comprise, from the N-terminus, the amino acid sequence of residue 512 toone of residues 514-521 of a human immunodeficiency virus type 1 (HIV-1)Envelope (Env) protein (according to the HXB2 numbering system). In someembodiments, the fusion proteins in the self-assembling proteinnanoparticle carrier further comprise a heterologous T-cell helperepitope. The immunogenic conjugate can be used elicit an immune responseto HIV-1 Env in a subject.

Immunogenic compositions including a disclosed immunogenic conjugate arealso provided. The composition may be a pharmaceutical compositionsuitable for administration to a subject, and may also be contained in aunit dosage form. The composition can further include an adjuvant.

Methods of generating an immune response to HIV-1 Env protein in asubject are disclosed, as are methods of treating, inhibiting orpreventing an HIV-1 infection in a subject. In such methods a subject,such as a human subject, is administered an effective amount of adisclosed immunogenic conjugate to elicit the immune response. Inseveral embodiments, the method comprises a prime-boost immunizationprotocol, where a disclosed immunogenic conjugate is used for the primeimmunization. The subject can be, for example, a human subject at riskof or having an HIV-1 infection.

The foregoing and other features and advantages of this disclosure willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1I depict embodiments of the self-assembling proteinnanoparticle carrier disclosed herein conjugated (FIGS. 1C-1I) or not(FIGS. 1A and 1B) to HIV-1 Env fusion peptides. As shown in FIG. 1A, theself-assembling protein nanoparticle carrier is a multimer of fusionproteins, each including a self-assembling protein nanoparticle subunitfused to a heterologous carrier protein. In some embodiments, the fusionprotein can further include a T-cell helper epitope (FIG. 1B), which isthen included in the self-assembling protein nanoparticle carrier. Thelocation of the T-cell-helper epitope can be varied in the fusionprotein. FIGS. 1C-1I, the HIV-1 Env fusion peptides (FP) are conjugatedto the self-assembling protein nanoparticle carrier. The HIV-1 Envfusion peptides can be conjugated to any suitable aspect of theself-assembling protein nanoparticle carrier. In some instances,sulfosuccinimidyl (4-iodoacetyl)aminobenzoate (Sulfo-SIAB) conjugationchemistry is used to conjugate the HIV-1 Env fusion peptides to exposedlysine residues of the self-assembling protein nanoparticle carrier.FIGS. 1G-1I illustrate additional embodiments that further include atargeting moiety that targets the immune system in a subject to enhancethe immune response to the HIV-1 Env fusion peptide on the immunogenicconjugate. The depictions in FIGS. 1A-1I are for illustration purposesand are not drawn to scale and do not necessarily show the number orrelative location of self-assembling protein nanoparticle subunits,carrier proteins, HIV-1 Env fusion peptides, and T-cell helper epitopesthat are present in a disclosed immunogenic conjugate.

FIG. 2 shows a set of images illustrating structural differences betweenKLH nanoparticles and KLH subunits, and a graph presenting data showingthat immunization with KLH nanoparticles conjugated to FP8 peptide(AVGIGAVF, residues 1-8 of SEQ ID NO: 1) elicits a much greater immuneresponse to the HIV-1 Env trimer than immunization with KLH subunitconjugated to FP8 peptide.

FIGS. 3A-3C shows a nanoparticle carrier assembly through genetic fusionof LS nanoparticle subunit and rTT carrier. FIG. 3A. Schematic of thefusion protein used to produce genetically fused rTT-LS nanoparticle.FIG. 3B. SEC profile of purified rTT-LS nanoparticle. FIG. 3C. Electronmicrographs of genetically fused rTT-LS nanoparticle carrier showsparticle species.

FIG. 4 shows electron micrographs of genetically fused rTT-LSnanoparticle carrier with a IgG hinge linking the rTT and LS subunit.

FIG. 5 shows electron micrographs for another example of a geneticallyfused nanoparticle carrier, formed from subunits of H. influenzaeprotein D fused to phosphopantetheine adenylyltransferase nanoparticlesubunit fused to rTT (HiD-6CCQ-rTT). The sequence of the fusion proteinused to generate these nanoparticle carrier is provided as SEQ ID NO:179. The observed particles were generally consistent in size and shapewith the known phosphopantetheine adenylyltransferase crystal structure(PDB 6CCQ).

FIGS. 6A-6C shows a nanoparticle carrier assembled through isopeptidebond fusion of lumazine synthase nanoparticle subunit and rTT carrier.FIG. 6A. Schematic of the lumazine synthase-spytag and rTT-spycatcherfusion proteins used to produce isopeptide bond-fused rTT-LSnanoparticle. Subsequent to formation of the rTT-LS nanoparticle, HIV-1fusion peptide (FP8) was conjugated to the nanoparticle-carrier by a PEGlinker FIG. 6B. Coomassie stained SDS-PAGE shows the individual purifiedproteins. FIG. 6C. SEC profile of purified rTT-LS nanoparticle.

FIG. 7 is a series of electron micrograph images of the purifiedrTT-SpyC fusion protein, the LS-SpyT nanoparticle, the LS-SpyTnanoparticle joined to the rTT-SpyC fusion protein (LS-Spy-rTT), and theLS-SpyT nanoparticle joined to the rTT-SpyC fusion protein furtherconjugated to HIV-1 Env fusion peptide FP8v1 by a PEG linker(LS-Spy-rTT-FP8v1/PEG2).

FIG. 8 shows results of isothermal calorimetry assays to determine thenumber of HIV-1 Env fusion peptides conjugated to monomeric rTT (FP-rTT)compared to the number of HIV-1 Env fusion peptides conjugated to theLS-SpyT nanoparticle joined to the rTT-SpyC fusion protein(LS-Spy-rTT-FP8v1/PEG2 nanoparticle carrier). The results show that eachFP-rTT monomer entity has six competent VRC34 Fab binding sites, whereaseach LS-Spy-rTT-FP8v1/PEG2 nanoparticle carrier has 152-402 competentVRC34.01 Fab binding sites.

FIG. 9 depicts an immunization protocol used to assess theLS-Spy-rTT-FP8v1/PEG2 nanoparticle carrier. For the first threeimmunizations (weeks 0, 3, and 6), mice received a 25 μg dose of eitherFP8v1-rTT monomer (Groups 1 and 2) or LS-Spy-rTT-FP8v1/PEG2 nanoparticlecarrier (Groups 3 and 4). For the following three immunizations, micereceived a 25 μg dose of either BG505 DS-SOSIP trimer (Groups 1 and 3)or the BG505 DS-SOSIP trimer conjugated to a lumazine synthasenanoparticle (Groups 2 and 4). Adjuplex was used as adjuvant for eachimmunization. Blood was drawn at weeks 0, 2, 5, 8, 11, 14, and 17.

FIGS. 10A-10C show binding and neutralization characteristics for serafrom FP-immunized mice. FIG. 10A. Week 2 and Week 5 sera was assessedfor FP binding by octet binding assay. FIG. 10B. Week, 2, 5, and 8 serawas assessed for BG505 trimer binding by ELISA. FIG. 10C. Week 17 serawas assessed for neutralization of BG505 virus with a mutation to removeglycan 611, as this viral variant is more sensitive to fusionpeptide-directed antibodies (Kong et al. Science 352, 828-833, 2016).

FIG. 11 shows a SDS-PAGE gel illustrating purification of an encapsulinnanoparticle subunit fused to a spytag. The encapsulin subunit includesG53C-R94C mutations to introduce a disulfide bond that stabilizesnanoparticles formed for the subunit.

FIG. 12 shows a SDS-PAGE gel illustrating purification of an encapsulinnanoparticle subunit fused to a spytag (EN-spytag), rTT carrier fused toa spycatcher moiety (rTT-spyC), and the encapsulin-rTT fusion (rTT-EN)formed from these two molecules. The encapsulin subunit includesG53C-R94C mutations to introduce a disulfide bond that stabilizesnanoparticles formed for the subunit.

FIG. 13 shows a series of electron micrograph images of the purifiedencapsulin-spytag, rTT-spy-encapsulin fusion, andFP8v1-rTT-spy-encapsulin.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt” (˜1.5 MB), which wascreated on Sep. 22, 2019, which is incorporated by reference herein.

DETAILED DESCRIPTION

As the HIV-1 pandemic continues to infect millions of people each year,the need for an effective vaccine increases. However, the development ofsuch a vaccine has been stymied due to the difficulty in developing animmunogen capable of eliciting broadly neutralizing antibodies. Thecurrent disclosure meets these needs.

One of the major hurdles to the construction of an effective HIV-1vaccine is focusing the immune response to regions of HIV proteins whichmostly produce broadly neutralizing antibodies. As disclosed herein, aseries of immunogens that elicit immune responses to the HIV-1 Envfusion peptide has been constructed. Such molecules have utility as bothpotential vaccines for HIV and as diagnostic molecules (for example, todetect and quantify target antibodies in a polyclonal serum response).

I. SUMMARY OF TERMS

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” It isfurther to be understood that any and all base sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescriptive purposes, unless otherwise indicated. Although many methodsand materials similar or equivalent to those described herein can beused, particular suitable methods and materials are described herein. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting. To facilitatereview of the various embodiments, the following explanations of termsare provided:

Adjuvant: A vehicle used to enhance antigenicity. In some embodiments,an adjuvant can include a suspension of minerals (alum, aluminumhydroxide, or phosphate) on which antigen is adsorbed; or water-in-oilemulsion, for example, in which antigen solution is emulsified inmineral oil (Freund incomplete adjuvant), sometimes with the inclusionof killed mycobacteria (Freund's complete adjuvant) to further enhanceantigenicity (inhibits degradation of antigen and/or causes influx ofmacrophages). In some embodiments, the adjuvant used in a disclosedimmunogenic composition is a combination of lecithin and carbomerhomopolymer (such as the ADJUPLEX™ adjuvant available from AdvancedBioAdjuvants, LLC, see also Wegmann, Clin Vaccine Immunol, 22(9):1004-1012, 2015). Additional adjuvants for use in the disclosedimmunogenic compositions include the QS21 purified plant extract, MatrixM, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides(such as those including a CpG motif) can also be used as adjuvants.Adjuvants include biological molecules (a “biological adjuvant”), suchas costimulatory molecules. Exemplary adjuvants include IL-2, RANTES,GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL andtoll-like receptor (TLR) agonists, such as TLR-9 agonists. Additionaldescription of adjuvants can be found, for example, in Singh (ed.)Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007).Adjuvants can be used in combination with the disclosed immunogens.

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition (such as a compositionincluding a disclosed immunogen) is administered by introducing thecomposition into a vein of the subject. Exemplary routes ofadministration include, but are not limited to, oral, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of an amino acid in apolypeptide with one or more different amino acids.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous antigens, such as the disclosedHIV antigens. Examples of antigens include, but are not limited to,polypeptides, peptides, lipids, polysaccharides, combinations thereof(such as glycopeptides) and nucleic acids containing antigenicdeterminants, such as those recognized by an immune cell. A vaccineantigen is an antigen that, when administered to a subject, elicits aprophylactic or therapeutic immune response in the subject.

Carrier protein: An immunogenic protein to which an antigen can belinked. When linked to a carrier, the antigen may become moreimmunogenic. Carriers are chosen to increase the immunogenicity of theantigen and/or to elicit antibodies against the carrier which arediagnostically, analytically, and/or therapeutically beneficial. Usefulcarrier proteins include polymeric carriers, which can be natural (forexample, proteins from bacteria or viruses), semi-synthetic or syntheticmaterials containing one or more functional groups to which a reactantmoiety can be attached.

Conjugated: A first moiety joined to a second moiety by a covalent bond.For example, a peptide (such as an HIV-1 Env fusion peptide) joined to acarrier (such as a self-assembling protein nanoparticle carrier asdescribed herein) by a chemical linker (such as a Sulfo-SIAB linker).

Conservative variant: “Conservative” amino acid substitutions are thosesubstitutions or deletions that do not substantially affect or decreasea function of a protein, such as the ability of the protein to elicit animmune response when administered to a subject. The term conservativeamino acid substitution also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid. Furthermore,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids (forinstance less than 5%, in some embodiments less than 1%) in an encodedsequence are conservative variations where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.

The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity orfunction of the recombinant Env protein, such as the ability to elicitan immune response when administered to a subject. For instance, if anamino acid residue is essential for a function of the protein, even anotherwise conservative substitution may disrupt that activity. Thus, aconservative substitution does not alter the basic function of a proteinof interest.

Consists essentially of and Consists Of: A polypeptide comprising anamino acid sequence that consists essentially of a specified amino acidsequence does not include any additional amino acid residues. However,the residues in the polypeptide can be modified to include non-peptidecomponents, such as labels (for example, fluorescent, radioactive, orsolid particle labels), sugars or lipids, and the N- or C-terminus ofthe polypeptide can be joined (for example, by peptide bond) toheterologous amino acids, such as a cysteine (or other) residue in thecontext of a linker for conjugation chemistry. A polypeptide thatconsists of a specified amino acid sequence does not include anyadditional amino acid residues, nor does it include additionalbiological components, such as nucleic acids lipids, sugars, nor does itinclude labels. However, the N- or C-terminus of the polypeptide can bejoined (for example, by peptide bond) to heterologous amino acids, suchas a peptide tag, or a cysteine (or other) residue in the context of alinker for conjugation chemistry.

A polypeptide that consists or consists essentially of a specified aminoacid sequence can be glycosylated or have an amide modification. Apolypeptide that consists of or consists essentially of a particularamino acid sequence can be linked via its N- or C-terminus to aheterologous polypeptide, such as in the case of a fusion proteincontaining a first polypeptide consisting or a first sequence that islinked (via peptide bond) to a heterologous polypeptide consisting of asecond sequence. In another example, the N- or C-terminus of apolypeptide that consists of or consists essentially of a particularamino acid sequence can be linked to a peptide linker (via peptide bond)that is further linked to one or more additional heterologouspolypeptides. In a further example, the N- or C-terminus of apolypeptide that consists of or consists essentially of a particularamino acid sequence can be linked to one or more amino acid residuesthat facilitate further modification or manipulation of the polypeptide.

Control: A reference standard. In some embodiments, the control is anegative control sample obtained from a healthy patient. In otherembodiments, the control is a positive control sample obtained from apatient diagnosed with HIV-1 infection. In still other embodiments, thecontrol is a historical control or standard reference value or range ofvalues (such as a previously tested control sample, such as a group ofHIV-1 patients with known prognosis or outcome, or group of samples thatrepresent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example, a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Covalent bond: An interatomic bond between two atoms, characterized bythe sharing of one or more pairs of electrons by the atoms. The terms“covalently bound” or “covalently linked” refer to making two separatemolecules into one contiguous molecule. The terms include reference toconjugating an antigen (such as an HIV-1 Env fusion peptide) eitherdirectly or indirectly to a carrier molecule, for example indirectlywith an intervening linker molecule.

Effective amount: An amount of agent, such as an immunogen, that issufficient to generate a desired response, such as an immune response ina subject. It is understood that to obtain a protective immune responseagainst an antigen of interest can require multiple administrations of adisclosed immunogen, and/or administration of a disclosed immunogen asthe “prime” in a prime boost protocol wherein the boost immunogen can bedifferent from the prime immunogen. Accordingly, an effective amount ofa disclosed immunogen can be the amount of the immunogen sufficient toelicit a priming immune response in a subject that can be subsequentlyboosted with the same or a different immunogen to generate a protectiveimmune response.

In one example, a desired response is to induce an immune response thatinhibits or prevents HIV-1 infection. The HIV-1 infected cells do notneed to be completely eliminated or prevented for the composition to beeffective. For example, administration of an effective amount of theimmunogen can induce an immune response that decreases the number ofHIV-1 infected cells (or prevents the infection of cells) by a desiredamount, for example, by at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or even at least100% (elimination or prevention of detectable HIV-1 infected cells), ascompared to the number of HIV-1 infected cells in the absence of theimmunization.

Expression: Transcription or translation of a nucleic acid sequence. Forexample, a gene is expressed when its DNA is transcribed into an RNA orRNA fragment, which in some examples is processed to become mRNA. A genemay also be expressed when its mRNA is translated into an amino acidsequence, such as a protein or a protein fragment. In a particularexample, a heterologous gene is expressed when it is transcribed into anRNA. In another example, a heterologous gene is expressed when its RNAis translated into an amino acid sequence. The term “expression” is usedherein to denote either transcription or translation. Regulation ofexpression can include controls on transcription, translation, RNAtransport and processing, degradation of intermediary molecules such asmRNA, or through activation, inactivation, compartmentalization ordegradation of specific protein molecules after they are produced.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (ATG) in front of a protein-encoding gene, splicing signal forintrons, maintenance of the correct reading frame of that gene to permitproper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Fusion protein: A single polypeptide chain including the sequence of twoor more heterologous proteins, often linked by a peptide linker.Reference to a first protein “fused” to a second protein indicates thatthe first and second proteins are contained within a single contiguouspolypeptide chain. The first and second protein may be directly linked(for example, the C-terminus of the first protein is linked to theN-terminus of the second protein by a peptide bond), or indirectlylinked (for example, the C-terminus of the first protein is directlylinked to the N-terminus of a peptide linker by a peptide bond, and theC-terminus of the peptide linker is directly linked to the N-terminus ofthe second protein by a peptide bond).

Heterologous: Originating from a different genetic source.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Human Immunodeficiency Virus Type 1 (HIV-1): A retrovirus that causesimmunosuppression in humans (HIV-1 disease), and leads to a diseasecomplex known as the acquired immunodeficiency syndrome (AIDS). “HIV-1disease” refers to a well-recognized constellation of signs and symptoms(including the development of opportunistic infections) in persons whoare infected by an HIV-1 virus, as determined by antibody or westernblot studies. Laboratory findings associated with this disease include aprogressive decline in T cells. Related viruses that are used as animalmodels include simian immunodeficiency virus (SIV), and felineimmunodeficiency virus (FIV). Treatment of HIV-1 with HAART has beeneffective in reducing the viral burden and ameliorating the effects ofHIV-1 infection in infected individuals.

HIV-1 envelope protein (Env): The HIV-1 Env protein is initiallysynthesized as a precursor protein of 845-870 amino acids in size.Individual precursor polypeptides form a homotrimer and undergoglycosylation within the Golgi apparatus as well as processing to removethe signal peptide, and cleavage by a cellular protease betweenapproximately positions 511/512 to generate separate gp120 and gp41polypeptide chains, which remain associated as gp120-gp41 protomerswithin the homotrimer. The ectodomain (that is, the extracellularportion) of the HIV-1 Env trimer undergoes several structuralrearrangements from a prefusion mature (cleaved) closed conformationthat evades antibody recognition, through intermediate conformationsthat bind to receptors CD4 and co-receptor (either CCR5 or CXCR4), to apostfusion conformation. The HIV-1 Env ectodomain comprises the gp120protein (approximately HIV-1 Env positions 31-511) and the gp41ectodomain (approximately HIV-1 Env positions 512-644). An HIV-1 Envectodomain trimer comprises a protein complex of three HIV-1 Envectodomains. As used herein “HIV-1 Env ectodomain trimer” includes bothsoluble trimers (that is, trimers without gp41 transmembrane domain orcytoplasmic tail) and membrane anchored trimers (for example, trimersincluding a full-length gp41).

Mature gp120 includes approximately HIV-1 Env residues 31-511, containsmost of the external, surface-exposed, domains of the HIV-1 Env trimer,and it is gp120 which binds both to cellular CD4 receptors and tocellular chemokine receptors (such as CCR5). A mature gp120 polypeptideis an extracellular polypeptide that interacts with the gp41 ectodomainto form an HIV-1 Env protomer that trimerizes to form the HIV-1 Envectodomain trimer. The mature gp120 wild-type polypeptide is heavilyN-glycosylated, giving rise to an apparent molecular weight of 120 kD.Native gp120 includes five conserved regions (C1-05) and five regions ofhigh variability (V1-V5).

Mature gp41 includes approximately HIV-1 Env residues 512-860, andincludes cytosolic-, transmembrane-, and ecto-domains. The gp41ectodomain (including approximately HIV-1 Env residues 512-644) caninteract with gp120 to form an HIV-1 Env protomer that trimerizes toform the HIV-1 Env trimer. The HIV-1 Env fusion peptide is located atthe N-terminus of gp41. Prior use of the HIV-1 Env fusion peptide forimmunization (e.g., as described in Dingens et al, Plos Pathog., 14(7),e1007159, 2018; and Xu et al., Nat. Med., 24(6):857-867, 2018, each ofwhich is incorporated by reference herein) illustrated HIV-1 Env fusionpeptide-based immunization protocols.

The prefusion mature closed conformation of the HIV-1 Env ectodomaintrimer is a structural conformation adopted by HIV-1 Env ectodomaintrimer after cellular processing to a mature prefusion state withdistinct gp120 and gp41 polypeptide chains, and before specific bindingto the CD4 receptor. The three-dimensional structure of an exemplaryHIV-1 Env ectodomain trimer in the prefusion mature closed conformationis known (see, e.g., Pancera et al., Nature, 514:455-461, 2014). In theprefusion mature closed conformation, the HIV-1 Env ectodomain trimerincludes a V1V2 domain “cap” at its membrane distal apex, with the V1V2domain of each Env protomer in the trimer coming together at themembrane distal apex. At the membrane proximal aspect, the prefusionmature closed conformation of the HIV-1 Env ectodomain trimer includesdistinct α6 and α7 helices. CD4 binding causes changes in theconformation of the HIV-1 Env ectodomain trimer, including disruption ofthe V1V1 domain cap, which “opens” as each V1V2 domain moves outwardfrom the longitudinal axis of the Env trimer, and formation of the HR1helix, which includes both the α6 and α7 helices (which are no longerdistinct). These conformational changes bring the N-terminus of thefusion peptide within close proximity of the target cell membrane, andexpose “CD4-induced” epitopes (such as the 17b epitope) that are presentin the CD4-bound open conformation, but not the mature closedconformation, of the HIV-1 Env ectodomain trimer.

Unless context indicates otherwise, the numbering used in the disclosedHIV-1 Env proteins and fragments thereof (such as a gp120 and gp41) isrelative to the HXB2 numbering scheme as set forth in NumberingPositions in HIV Relative to HXB2CG Bette Korber et al., HumanRetroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acidand Amino Acid Sequences. Korber et al., Eds. Theoretical Biology andBiophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex.,which is incorporated by reference herein in its entirety. Forreference, the amino acid sequence of HIV-1 Env of HXB2 is set forth asSEQ ID NO: 154 (GENBANK® GI:1906382, incorporated by reference herein aspresent in the database on Jun. 20, 2014).

HXB2 (Clade B, SEQ ID NO: 13):MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQGLERILL

HIV-1 neutralizing antibody: An antibody that reduces the infectioustiter of HIV-1 by binding to HIV-1 Env protein and inhibiting HIV-1function. In some embodiments, neutralizing antibodies to HIV-1 caninhibit the infectivity of multiple strains of HIV-1, Teir-2 strain frommultiple clades of HIV-1. In some embodiments, a disclosed immunogen canbe administered to a subject to elicit an immune response that includesproduction of antibodies that specifically bind to the HIV-1 Env fusionpeptide and neutralize Teir-2 strains of HIV-1 from multiple HIV-1clades.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies. “Priming an immune response” refers to treatment of asubject with a “prime” immunogen to induce an immune response that issubsequently “boosted” with a boost immunogen. Together, the prime andboost immunizations produce the desired immune response in the subject.“Enhancing an immune response” refers to co-administration of anadjuvant and an immunogenic agent, wherein the adjuvant increases thedesired immune response to the immunogenic agent compared toadministration of the immunogenic agent to the subject in the absence ofthe adjuvant.

Immunogen: A protein or a portion thereof that is capable of inducing animmune response in a mammal, such as a mammal infected or at risk ofinfection with a pathogen.

Immunogenic composition: A composition comprising a disclosed immunogenthat elicits a measurable CTL response against the immunogen, or elicitsa measurable B cell response (such as production of antibodies) againstthe immunogen, when administered to a subject. For in vivo use, theimmunogenic composition will typically include the immunogen in apharmaceutically acceptable carrier and may also include other agents,such as an adjuvant.

Immunogenic conjugate: A composition including of at least twoheterologous molecules (such as an HIV-1 Env fusion peptide and acarrier, such as a self-assembling protein nanoparticle carrier)conjugated together. In a non-limiting example, a peptide (such asAVGIGAVF peptide, residues 1-8 of SEQ ID NO: 1) is linked to a proteincarrier by a linker including a heterologous cysteine residue fused tothe C-terminal residue of the peptide by peptide bond and aheterobifunctional moiety, wherein the heterobifunctional moiety islinked to a lysine residue on the carrier and the cysteine residue. Inthis example, the peptide is indirectly covalently linked to the carrierby the linker Immunogenic conjugates are conjugates that are useful foreliciting a specific immune response to a molecule in the conjugate in avertebrate. In some embodiments where the conjugate includes a viralantigen, the immune response is protective in that it enables thevertebrate animal to better resist infection from the virus from whichthe antigen is derived.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as acquired immunodeficiency syndrome (AIDS). “Treatment”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.The term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.Inhibiting a disease can include preventing or reducing the risk of thedisease, such as preventing or reducing the risk of viral infection. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, a reduction in the viral load, animprovement in the overall health or well-being of the subject, or byother parameters that are specific to the particular disease. A“prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Isolated: An “isolated” biological component has been substantiallyseparated or purified away from other biological components, such asother biological components in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA, RNA, and proteins.Proteins, peptides, nucleic acids, and viruses that have been “isolated”include those purified by standard purification methods. Isolated doesnot require absolute purity, and can include protein, peptide, nucleicacid, or virus molecules that are at least 50% isolated, such as atleast 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Linked: The term “linked” means joined together, either directly orindirectly. For example, a first moiety may be covalently ornoncovalently (e.g., electrostatically) linked to a second moiety. Thisincludes, but is not limited to, covalently bonding one molecule toanother molecule, noncovalently bonding one molecule to another (e.g.electrostatically bonding), non-covalently bonding one molecule toanother molecule by hydrogen bonding, non-covalently bonding onemolecule to another molecule by van der Waals forces, and any and allcombinations of such couplings. Indirect attachment is possible, such asby using a “linker”. In several embodiments, linked components areassociated in a chemical or physical manner so that the components arenot freely dispersible from one another, at least until contacting acell, such as an immune cell.

Linker: One or more molecules or groups of atoms positioned between twomoieties. Typically, linkers are bifunctional, i.e., the linker includesa functional group at each end, wherein the functional groups are usedto couple the linker to the two moieties. The two functional groups maybe the same, i.e., a homobifunctional linker, or different, i.e., aheterobifunctional linker. In several embodiments, a peptide linker canbe used to link the C-terminus of a first protein to the N-terminus of asecond protein. Non-limiting examples of peptide linkers includeglycine-serine peptide linkers, which are typically not more than 10amino acids in length. In a non-limiting example, a peptide (such asAVGIGAVF peptide, residues 1-8 of SEQ ID NO: 1) is linked to a proteincarrier by a linker including a heterologous cysteine residue fused tothe C-terminal residue of the peptide by peptide bond and aheterobifunctional moiety, wherein the heterobifunctional moiety islinked to a lysine residue on the carrier and the cysteine residue.

Nucleic acid molecule: A polymeric form of nucleotides, which mayinclude both sense and anti-sense strands of RNA, cDNA, genomic DNA, andsynthetic forms and mixed polymers of the above. A nucleotide refers toa ribonucleotide, deoxynucleotide or a modified form of either type ofnucleotide. The term “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide.” A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term includes single- and double-stranded forms of DNA. Apolynucleotide may include either or both naturally occurring andmodified nucleotides linked together by naturally occurring and/ornon-naturally occurring nucleotide linkages. “cDNA” refers to a DNA thatis complementary or identical to an mRNA, in either single stranded ordouble stranded form. “Encoding” refers to the inherent property ofspecific sequences of nucleotides in a polynucleotide, such as a gene, acDNA, or an mRNA, to serve as templates for synthesis of other polymersand macromolecules in biological processes having either a definedsequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a definedsequence of amino acids and the biological properties resultingtherefrom.

Pattern recognition receptor: A protein receptor expressed by cells ofthe immune system to identify pathogen-associated molecular patterns(PAMPS) as well as damage associated molecular patterns (DAMPs). PAMP orDAMP activation of pattern recognition receptors induces anintracellular signaling cascade resulting in the alteration of the hostcell's transcription profile to induce expression of pro-inflammatoryand pro-survival genes that enhance adaptive immunity Non-limitingexamples of pattern recognition receptors (PRRs) include Toll-likereceptors (TLR), Stimulator of Interferon Genes receptor (STING), C-typelectin receptors (CLR), RIG-I-like receptors (RLR), and NOD-likereceptors (NLR). In some embodiments, agonists of such patternrecognition receptors can be linked to a disclosed immunogenic conjugateto target the conjugate to pattern recognition receptor expressing cells(i.e., cells of the immune system) to enhance the immune response to theimmunogenic conjugate.

Toll-like receptors (TLRs) 1-13 are transmembrane PRRs that recognize adiverse range of PAMPs. TLRs can be divided into two broadcategories—those that are localized to the cell surface and those thatare localized to the endosomal lumen. TLRs that are present on the cellsurface are important in recognition of bacterial pathogens. TLRs thatare localized to the lumen of endosomes, such as TLRs 3, 7, 8, and 9,serve to recognize nucleic acids and are thus thought to be important inthe promotion of antiviral immune responses. TLR-7 and TLR-8 recognizessRNA. Several different imidazoquinoline compounds are known TLR-7/8agonists. TLR-9 recognizes unmethylateddeoxycytidylate-phosphate-deoxyguanylate (CpG) DNA, found primarily inbacteria.

The NOD-like receptors (NLRs) and the RIG-I-like receptors (RLRs) arelocalized to the cytoplasm. Non-limiting examples of RLRs include RIG-I,MDA5, and LGP2. There are 22 human NLRs that can be subdivided into thefive structurally related NLR families A, B, C, P, and X. All NLRs havethree domains: an N-terminal domain involved in signaling, anucleotide-binding NOD domain, and a C-terminal leucine rich region(LRR) important for ligand recognition. Non-limiting examples of NLRsinclude NALP3 and NOD2.

For more information on pattern recognition receptors, see Wales et al.,Biochem Soc Trans., 35:1501-1503, 2007.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example, sodiumacetate or sorbitan monolaurate. In particular embodiments, suitable foradministration to a subject the carrier may be sterile, and/or suspendedor otherwise contained in a unit dosage form containing one or moremeasured doses of the composition suitable to elicit the desiredanti-HIV-1 immune response. It may also be accompanied by medicationsfor its use for treatment purposes. The unit dosage form may be, forexample, in a sealed vial that contains sterile contents or a syringefor injection into a subject, or lyophilized for subsequentsolubilization and administration or in a solid or controlled releasedosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example, an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues.

Prime-boost immunization: An immunotherapy including administration ofmultiple immunogens over a period of time to elicit the desired immuneresponse.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring, for example, includes one ormore nucleic acid substitutions, deletions or insertions, and/or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination can beaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of isolated segments of nucleic acids, for example, bygenetic engineering techniques. A recombinant virus is one that includesa genome that includes a recombinant nucleic acid molecule.

A recombinant protein is one that has a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. In several embodiments, arecombinant protein is encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell, or into the genome of arecombinant virus.

Self-assembling protein nanoparticle: A multi-subunit protein-basednanoparticle formed from subunit monomers that self-assemble undersuitable conditions to form the nanoparticle (typically globular inshape). Non-limiting examples of self-assembling protein nanoparticlesinclude ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci.,12:5406-5421, 2011, incorporated by reference herein), encapsulinnanoparticles (see, e.g., Sutter et al., Nature Struct. and Mol. Biol.,15:939-947, 2008, incorporated by reference herein), Sulfur OxygenaseReductase (SOR) nanoparticles (see, e.g., Urich et al., Science,311:996-1000, 2006, incorporated by reference herein), lumazine synthasenanoparticles (see, e.g., Zhang et al., J. Mol. Biol., 306: 1099-1114,2001), and pyruvate dehydrogenase nanoparticles (see, e.g., Izard etal., PNAS 96: 1240-1245, 1999, incorporated by reference herein).Ferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenaseare monomeric proteins that self-assemble into a globular proteincomplexes that in some cases consists of 24, 60, 24, 60, and 60 proteinsubunits, respectively. In some examples, ferritin, encapsulin, SOR,lumazine synthase, or pyruvate dehydrogenase subunits are fused to adisclosed heterologous carrier protein (such as an rTT, CRM197, or HiDcarrier protein) and self-assembled into a protein nanoparticlepresenting the carrier protein, which can subsequently be conjugated toHIV-1 Env fusion proteins to generate an immunogenic conjugate to elicitor prime an immune response to HIV-1 Env in a subject.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity; the higher the percentage, themore similar the two sequences are. Homologs, orthologs, or variants ofa polypeptide will possess a relatively high degree of sequence identitywhen aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Variants of a polypeptide are typically characterized by possession ofat least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over thefull length alignment with the amino acid sequence of interest. Proteinswith even greater similarity to the reference sequences will showincreasing percentage identities when assessed by this method, such asat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity. When less than the entire sequence isbeing compared for sequence identity, homologs and variants willtypically possess at least 80% sequence identity over short windows of10-20 amino acids, and may possess sequence identities of at least 85%or at least 90% or 95% depending on their similarity to the referencesequence. Methods for determining sequence identity over such shortwindows are available at the NCBI website on the internet.

As used herein, reference to “at least 90% identity” (or similarlanguage) refers to “at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or even 100% identity” to a specified referencesequence.

Signal Peptide: A short amino acid sequence (e.g., approximately 18-30amino acids in length) that directs newly synthesized secretory ormembrane proteins to and through membranes (for example, the endoplasmicreticulum membrane). Signal peptides are typically located at theN-terminus of a polypeptide and are removed by signal peptidases afterthe polypeptide has crossed the membrane. Signal peptide sequencestypically contain three common structural features: an N-terminal polarbasic region (n-region), a hydrophobic core, and a hydrophilicc-region). An exemplary signal peptide sequence is set forth asMDSKGSSQKGSRLLLLLVVSNLLLPQGVVA (SEQ ID NO: 220).

Specifically bind: When referring to the formation of anantibody:antigen protein complex, or a protein:protein complex, refersto a binding reaction which determines the presence of a target protein,peptide, or polysaccharide (for example, a glycoprotein), in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated conditions, a particular antibody or proteinbinds preferentially to a particular target protein, peptide orpolysaccharide (such as an antigen present on the surface of a pathogen,for example, gp120) and does not bind in a significant amount to otherproteins or polysaccharides present in the sample or subject. Specificbinding can be determined by standard methods. A first protein orantibody specifically binds to a target protein when the interaction hasa K_(D) of less than 10⁻⁶ Molar, such as less than 10⁻⁷ Molar, less than10⁻⁸ Molar, less than 10⁻⁹, or even less than 10⁻¹⁰ Molar.

Subject: Living multicellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In a particular example, the subject is a newborn infant. In anadditional example, a subject is selected that is in need of inhibitingof an HIV-1 infection. For example, the subject is either uninfected andat risk of HIV-1 infection or is infected in need of treatment.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity.

Vaccine: A pharmaceutical composition that elicits a prophylactic ortherapeutic immune response in a subject. In some cases, the immuneresponse is a protective immune response. Typically, a vaccine elicitsan antigen-specific immune response to an antigen of a pathogen, forexample a viral pathogen, or to a cellular constituent correlated with apathological condition. A vaccine may include a polynucleotide (such asa nucleic acid encoding a disclosed antigen), a peptide or polypeptide(such as a disclosed antigen), a virus, a cell or one or more cellularconstituents. In one specific, non-limiting example, a vaccine reducesthe severity of the symptoms associated with HIV-1 infection and/ordecreases the viral load compared to a control. In another non-limitingexample, a vaccine reduces HIV-1 infection compared to a control.

VRC34: An antibody that binds to the fusion peptide of HIV-1 anyneutralizing HIV-1 infection. VRC34. Unless context indicates otherwise,“VRC34” refers to the VRC34.01 antibody disclosed by Kong et al.(Science, 352, 828-833, 2016). Sequences of the heavy and light chainvariable regions of the VRC34.01 antibody are available, for example, asGenBank Accession Nos. ANF29805.1 and ANF29798.1, respectively, each ofwhich is incorporated by reference herein. The VRC34 antibody can beused to assess the antigenicity fo the disclosed immunogenic conjugatesof HIV-1 Env fusion peptides conjugated to a self-assembling proteinnanoparticle carrier.

II. IMMUNOGENIC CONJUGATES

Immunogenic conjugates are provided herein that include HIV-1 Env fusionpeptides conjugated to a self-assembling protein nanoparticle carrier.In several embodiments, the immunogenic conjugates can be used togenerate a neutralizing immune response to HIV-1 in a subject, forexample, to treat or prevent an HIV-1 infection in the subject. Theimmunogenic conjugate provides a multivalent platform with superiorbinding capability for engaging HIV-1 Env fusion peptide-directedbroadly neutralizing antibodies and can be used, for example, to primean immune response in a subject that targets the HIV-1 Env fusionpeptide epitope. The components of the immunogenic conjugate arediscussed in more detail below.

A. Self-Assembling Protein Nanoparticle Carrier

The immunogenic conjugates provided herein include HIV-1 Env fusionpeptides conjugated to a self-assembling protein nanoparticle carrier.The self-assembling protein nanoparticle carrier is formed from amultimer of fusion proteins that each include a self-assembling proteinnanoparticle subunit fused to a heterologous carrier protein. Thesubunit and the carrier protein can be directly fused (the C-terminus ofone is linked by peptide bond to the N-terminus of the other) orindirectly fused via a peptide linker. Following expression of thefusion proteins (typically in a cellular system where the fusionproteins are secreted into the supernatant), the self-assembling proteinnanoparticle subunits of the fusion proteins self-assemble undersuitable conditions into the protein nanoparticle, forming a proteincomplex containing the protein nanoparticle and displaying theheterologous carrier proteins (at least one of which is fused to eachself-assembling protein nanoparticle subunit). This protein complex isthe self-assembling protein nanoparticle carrier to which HIV-1 Envfusion peptides are conjugated to form an immunogenic conjugate of thepresent disclosure.

1. Self-Assembling Protein Nanoparticle Subunit

The fusion proteins of the self-assembling protein nanoparticle carrierinclude a self-assembling protein nanoparticle subunit fused to aheterologous carrier protein. The self-assembling protein nanoparticlesubunit is a monomer of a self-assembling protein nanoparticle, or afragment of such a monomer that retains the portion of the monomerrequired for self-assembly. Non-limiting examples of self-assemblingprotein nanoparticle subunits that can be included in the fusion proteinto form a self-assembling protein nanoparticle carrier include lumazinesynthase nanoparticle subunits, ferritin nanoparticle subunts,encapsulin nanoparticle subunits, Sulfur Oxygenase Reductase (SOR)nanoparticle subunits, Bacteriophage Q Beta Capsid protein (qbeta)subunits, Dihydrolipoyl transacetylase protein (e2p) subunits,Phosphopantetheine Adenylyltransferase (6ccq) subunits, GlutamateSynthase (1f52) subunits, Calcium/calmodulin dependent protein kinase Ha(CaMKIIa), C-terminal fragment (5U6Y) subunits, HIV capsidoligomerization domain subunits, Hexamer subunits, and T4 fibritinFoldon domain (Fd) subunits. In a preferred embodiment, theself-assembling protein nanoparticle subunit included in the fusionprotein is a ferritin subunit. In another preferred embodiment, theself-assembling protein nanoparticle subunit included in the fusionprotein is a lumazine synthase subunit.

a. Ferritin

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to aferritin subunit to construct a self-assembling ferritin nanoparticlecarrier including a ferritin nanoparticle fused to a plurality of theheterologous carrier proteins. Ferritin nanoparticles and their use forimmunization purposes (e.g., for immunization against influenzaantigens) have been disclosed, for example, in Kanekiyo et al. (Nature,499:102-106, 2013, incorporated by reference herein in its entirety).Ferritin is a globular protein that is found in all animals, bacteria,and plants, and which acts primarily to control the rate and location ofpolynuclear Fe(III)₂O₃ formation through the transportation of hydratediron ions and protons to and from a mineralized core. Ferritinnanoparticles are formed from 24 copies of the ferritin subunit. Theglobular form of the ferritin nanoparticle is made up of monomericsubunits. Non-limiting examples of the sequence of self-assemblingferritin subunits for use in the embodiments provided herein include:

(SEQ ID NO: 14) DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKS(SEQ ID NO: 15) DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPHHKFHGLTHIFHKAYHHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKS(SEQ ID NO: 16) DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEEKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKS(SEQ ID NO: 17) DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEEKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKS(SEQ ID NO: 18) DIIKLLNEQVNKEMDSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEEKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY VKGIAKSRKSand the following two sequences which include C-terminal truncations:

(SEQ ID NO: 19) DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIG (SEQ ID NO: 20)DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEN

Additional ferritin subunits are provided with one or more cysteinesubstitutions to introduce non-native disulfide bond(s) that stabilizethe ferritin nanoparticle formed from the self-assembled subunits. Asused herein, a non-native disulfide bond introduced into aself-assembling protein nanoparticle subunit that “stabilizes” thenanoparticle formed from oligomerization of the subunit increasesretention of the assembled nanoparticle compared to a controlnanoparticle formed from subunits lacking the disulfide bond. The“stabilization” of the nanoparticle can be, for example, an increase inresistance to disassembly of the subunits compared to a correspondingnative subunit sequence. Non-limiting examples of ferritin subunits areprovided with one or more cysteine substitutions to introduce non-nativedisulfide bond(s) that stabilize the ferritin nanoparticle formed fromthe self-assembled subunits include:

Ferr_Hp_DS01 (SEQ ID NO: 258)MLSKDIIKLLNEQVNKEMQSSNLYMSMSCWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPCQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHCTFNFLQWYVAEQCEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS02 (SEQ ID NO: 259)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTGCISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHCTFNFLQWYVAEQCEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS03 (SEQ ID NO: 260)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTCISCPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHCTFNFLQWYVAEQCEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS04 (SEQ ID NO: 261)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSCSAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHCTFNFLQWYVAEQCEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS05 (SEQ ID NO: 262)MLSKDIIKLLNEQVNKEMQSSNLYMSMSCWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNCNNVPCQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEECLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS06 (SEQ ID NO: 263)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNONNVPVQLTGCISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEECLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS07 (SEQ ID NO: 264)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNCNNVPVQLTCISPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEELFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS08 (SEQ ID NO: 265)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNCNNVPVQLTSCSAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEECLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS09 (SEQ ID NO: 266)MLSKDIIKLLNEQVNKEMQSSNLYMSMSCWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNI+32AIKSKDCATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS10 (SEQ ID NO: 267)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTGCISAPEHKFEGLTQIFQKAYEHEQHISESINNICDHAIKSKDCATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS11 (SEQ ID NO: 268)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTCISPEHKFEGLTQIFQKAYEHEQHISESINNICDHAIKSKDATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_Hp_DS12 (SEQ ID NO: 269)MLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSCSAPEHKFEGLTQIFQKAYEHEQHISESINNICDHAIKSKDCATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr_pf_DS01 (SEQ ID NO: 270)MLSERMLKALNDQLNRELYSAYLYFAMACYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYCRNGRELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS02 (SEQ ID NO: 271)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYCRNGRVELDCIPCPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS03 (SEQ ID NO: 272)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYORNGRVELDECPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS04 (SEQ ID NO: 273)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYCRNGRVELDCIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS05 (SEQ ID NO: 274)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYCRNGRVELDGCIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS06 (SEQ ID NO: 275)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYORNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGGC Ferr_pf_DS07 (SEQ ID NO: 276)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYCRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEECSVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGGGWC Ferr_pf_DS08 (SEQ ID NO: 277)MLSERMLKALNDQLNRELYSAYLYFAMACYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRCELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFCNEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS09 (SEQ ID NO: 278)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDCIPCPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFCNEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS10 (SEQ ID NO: 279)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDECPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFCNEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS11 (SEQ ID NO: 280)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDCIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFCNEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS12 (SEQ ID NO: 281)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDGCIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFONEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE Ferr_pf_DS13 (SEQ ID NO: 282)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFCNEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGGGC Ferr_pf_DS14 (SEQ ID NO: 283)MLSERMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDCSTRAFLEWFCNEQVEEEASVKKILDKLKFAKDSPQILFMLDKELSARAPKLPGGGWC Ferr_Mt_DS01 (SEQ ID NO: 284)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVCIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLCRDLRVECPGVDTVRNQFDRPREALALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVCLMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL Ferr_Mt_DS02 (SEQ ID NO: 285)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLCRDLRVEIPGCDTVRNQFDRPREALALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVCLMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL Ferr_Mt_DS03 (SEQ ID NO: 286)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLCRDLRVCIPGVDTVRCQFDRPREALALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEWLMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL Ferr_Mt_DS04 (SEQ ID NO: 287)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLCRDLRVEIPGVDTVRNQFDRPREALALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVCLMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRC Ferr_Mt_DS05 (SEQ ID NO: 288)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLCRDLRVEIPGVDTVRNQFDRPREALALALDQERTVTDQVGRLTAVARDEGDFLGEQFMQWFLQEQIEEVCLMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRC Ferr_Mt_DS06 (SEQ ID NO: 289)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVCIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVECPGVDTVRNQFDRPREALALALDQERTVTDQVGRLCAVARDEGDCLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL Ferr_Mt_DS07 (SEQ ID NO: 290)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGCDTVRNQFDRPREALALALDQERTVTDQVGRLOAVARDEGDLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL Ferr_Mt_DS08 (SEQ ID NO: 291)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVCIPGVDTVRCQFDRPREALALALDQERTVTDQVGRLCAVARDEGDCLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRL Ferr_Mt_DS09 (SEQ ID NO: 292)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPREALALALDQERTVTDQVGRLAVARDEGDOLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGR Ferr_Mt_DS10 (SEQ ID NO: 293)MTEYEGPKTKFHALMQEQIHNEFTAAQQYVAIAVYFDSEDLPQLAKHFYSQAVEERNHAMMLVQHLLDRDLRVEIPGVDTVRNQFDRPREALALALDQERTVTDQVGRLCAVARDEGDCLGEQFMQWFLQEQIEEVALMATLVRVADRAGANLFELENFVAREVDVAPAASGAPHAAGGRG Ferr_ec_DS01 (SEQ ID NO: 294)MLKPEMIEKLNEQMNLELYSSLLYQQMSAW+32HTFEGAAAFLRRHAQEEMTHMQRLFDYLCDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELCHAAMTNQDCPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS02 (SEQ ID NO: 295)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINCVECPFAEYSSLDELFQETYKHEQLITQKINELHAAMTNQDCPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS03 (SEQ ID NO: 296)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTCESPFAEYSSLDELFQETYKHEQLITQKINELCHAAMTNQDCPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS04 (SEQ ID NO: 297)MLKPEMIEKLNEQMNLELYSSLLYQQMSAW+32HTFEGAAAFLRRHAQEEMTHMQRLFDYLCDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYQTFNFLQWYCSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS05 (SEQ ID NO: 298)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINCVECPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYCTFNFLQWYSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS06 (SEQ ID NO: 299)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTCESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYCTFNFLQWWSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS07 (SEQ ID NO: 300)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCCYHTFEGAAAFLRRHAQEEMTHMQRLFDYLCDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQD+32FNFLQWYVSEQEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS08 (SEQ ID NO: 301)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCCYHTFEGAAAFLRRHAQEEMTHMQRLFDYLQDTGNLPRINTVESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYQTFNFLQWYVSEQCGEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS09 (SEQ ID NO: 302)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINCVECPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYCTFNFLQWYVSEQCEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS10 (SEQ ID NO: 303)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINCVECPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYCTFNFLQWYVSEQCGEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_ec_DS11 (SEQ ID NO: 304)MLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTQESPFAEYSSLDELFQETYKHEQLITQKINELAHAAMTNQDYCTFNFLQWYVSEQCGEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN Ferr_frog_DS01 (SEQ ID NO: 305)MVSQCRQNYHSDCEAAVNRMLNLELYASYTYSSMYCFFDRDDVALHNVAEFFKEHSHEEREHAEKFMKYQNKRGGRCVLQDIKKPERDEWGNTLEAMQAALQLEKTVNQALLDLHKLATDKVDPHLCDFLESEYLEEQVKDIKRICDFITNLKRLGLPENGMGEYLFDKHSVKESS

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a ferritin subunit including an amino acid sequence atleast 80% (such as at least 85%, at least 90%, at least 95%, or at least97%) identical to amino acid sequence set forth as any one of SEQ IDNOs: 14-20 or 258-305.

In additional embodiments, any of the disclosed heterologous carrierproteins can be linked to an insect ferritin subunit to construct theself-assembling ferritin nanoparticle carrier including the ferritinnanoparticle fused to the plurality of the heterologous carrierproteins. Insect ferritin protein nanopartciles and their use andproduction are described, for example, in PCT. Pub. No. WO 2018/005558,which is incorporated by reference herein. Unlike bacterial ferritin,insect ferritin includes twelve copies of two different subunits (termedheavy and light chains; 24 subunits total). The insect ferritin heavychains trimerize and the insect ferritin light chains trimerize (formingfour trimers of heavy chains and four trimers of light chains) andself-assemble into the globular nanoparticle. In several embodiments,each insect ferritin heavy chain includes an N-terminal fusion to afirst heterologous carrier protein, and each insect ferritin light chainincludes an N-terminal fusion to a second heterologous carrier protein.This allows for display of two diverse carrier proteins on the sameferritin nanoparticle.

In several embodiments, the insect ferritin heavy and light chains canbe from the Lepidoptera order of insects, such as ferritin heavy andlight chains from Trichoplusia (such as Trichoplusia ni), or ferritinheavy and light chains from Manduca. Exemplary ferritin heavy and lightchain amino acid sequences for Trichoplusia ni and Manduca proteins areprovided below:

Exemplary insect ferritin heavy and light chain sequences withN-terminal truncations that can be included in the fusion protein areset forth below:

Trichoplusia ni ferritin heavy chain with18-aa N-terminal truncation (nt19) (SEQ ID NO: 21)RSCRNSMRQQIQMEVGASLQYLAMGAHFSKDVVNRPGFAQLFFDAASEEREHAMKLIEYLLMRGELTNDVSSLLQVRPPTRSSWKGGVEALEHALSMESDVTKSIRNVIKACEDDSEFNDYHLVDYLTGDFLEEQYKGQRDLAGKASTLKKLMDRHEALGEFIFDKKLLGIDV Trichoplusia ni ferritin light chain with29-aa N-terminal truncation (nt30) (SEQ ID NO: 22)EYGSHGNVATELQAYAKLHLERSYDYLLSAAYFNNYQTNRAGFSKLFKKLSDEAWSKTIDIIKHVTKRGDKMNFDQHSTMKTERKNYTAENHELEALAKALDTQKELAERAFYIHREATRNSQHLHDPEIAQYLEEEFIEDHAEKIRTLAGHTSDLKKFITANNGHDLSLALYVFDEYLQKTVManduca ferritin heavy chain with 38-aa truncation (nt39)(SEQ ID NO: 23) RSCRDSMRRQIQMEVGASLQYLAMGAHFSKDKINRPGFAKLFFDAAGEEREHAMKLIEYLLMRGELTNDVTSLIQVRAPQRNKWEGGVDALEHALKMESDVTKSIRTVIKACEDDPEFNDYHLVDYLTGEFLEEQYKGQRDLAGKASTLKKMLDRNSALGEFIFDKKLMGMDI Manduca ferritin light chain with 48-aaN-terminal truncation (nt49) (SEQ ID NO: 24)EYGHHGNVAKEMQAYAALHLERSYEYLLSSSYFNNYQTNRAGFSKLFRKLSDDAWEKTIDLIKHITMRGDEMNFAQRSTQKSVDRKNYTVELHELESLAKALDTQKELAERAFFIHREATRNSQHLHDPEVAQYLEEEFIEDHAKTIRNLAGHTTDLKRFVSGDNGQDLSLALYVFDEYLQKTV

In some embodiments, the insect ferritin heavy chain can be aTrichoplusia ni ferritin heavy chain with an 18 amino acid N-terminaltruncation and the insect ferritin light chain can be a Trichoplusia niferritin light chain with a 29 amino acid N-terminal truncation. Forexample, the insect ferritin heavy chain comprises an amino acidsequence at least 90% identical to SEQ ID NO: 21, and the insectferritin light chain comprises an amino acid sequence at least 90%identical SEQ ID NO: 22 In some embodiments, the insect ferritin heavychain comprises an amino acid sequence set forth as SEQ ID NO: 21, andthe insect ferritin light chain comprises an amino acid sequence setforth as SEQ ID NO: 22.

In some embodiments, the insect ferritin heavy chain can be a Manducaferritin heavy chain with a 38 amino acid N-terminal truncation and theinsect ferritin light chain can be a Manduca ferritin light chain with a48 amino acid N-terminal truncation. For example, the insect ferritinheavy chain comprises an amino acid sequence at least 90% identical toSEQ ID NO: 23, and the insect ferritin light chain comprises an aminoacid sequence at least 90% identical to SEQ ID NO: 24. In someembodiments, the insect ferritin heavy chain comprises an amino acidsequence set forth as SEQ ID NO: 23, and the insect ferritin light chaincomprises an amino acid sequence set forth as SEQ ID NO: 24.

b. Lumazine Synthase (LS)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to alumazine synthase subunit to construct a self-assembling lumazinesynthase nanoparticle carrier including a lumazine synthase nanoparticlefused to a plurality of the heterologous carrier proteins. Lumazinesynthase nanoparticles are formed from 60 copies of the lumazinesynthase subunit.

The globular form of lumazine synthase nanoparticle is made up ofmonomeric subunits; non-limiting examples of the sequence of lumazinesynthase subunits are provided as:

(SEQ ID NO: 25) MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLF KSLR (SEQ ID NO: 26)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFK SLR (SEQ ID NO: 27)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFK SLR (SEQ ID NO: 28)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFK SLR

Additional lumazine synthase subunits are provided with one or morecysteine substitutions to introduce non-native disulfide bond(s) thatstabilize the lumazine synthase nanoparticle formed from self-assembledsubunits. In some embodiments, the non-native disulfide bond(s) areintroduced with L121C-K131C, L121CG-K131C, L121GC-K131C, K7C-R40C,I3C-L50C, I82C-K131CG, E5C-R52C, or E95C-A101C substitutions, or acombination thereof (such as I3C-L50C and I82C-K131CG; E5C-R52C andI82C-K131CG; or E95C-A101C and I82C-K131CG). The residues numbering iswith reference to the lumazine synthase subunit set forth as SEQ ID NO:25. Non-limiting examples include:

LS-L121C-K131C (SEQ ID NO: 306)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLV RVPGSWEIPVAAGELARKEnIs AVIAIGVLIRGATPHFDYIASEVSKGLA DLSLELRKPITFGVITADT c EQAIERAGT cHGNKGWEAALSAIEMANLFK SLR LS-L121CG-K131C (SEQ ID NO: 307)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLV RVPGSWEIPVAAGELARKEnIs AVIAIGVLIRGATPHFDYIASEVSKGLA DLSLELRKPITFGVITADT cg EQAIERAGT cHGNKGWEAALSAIEMANLF KSLR LS-L121GC-K131C (SEQ ID NO: 308)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLV RVPGSWEIPVAAGELARKEnIs AVIAIGVLIRGATPHFDYIASEVSKGLA DLSLELRKPITFGVITADT gc EQAIERAGT cHGNKGWEAALSAIEMANLF KSLR LS-K7C-R40C (SEQ ID NO: 309) QIYEG cLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVcHGGREEDITLV RVPGSWEIPVAAGELARKE nIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFK SLRLS_Aq_DS01 (I3C-L50C, I82C-K131CG) (SEQ ID NO: 310)QCYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITCVRVPGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTCGHGNKGWEAALSAIEMANLF KSLRLS_Aq_DS02 (E5C-R52C, I82C-K131CG) (SEQ ID NO: 311)QIYCGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVCVPGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTCGHGNKGWEAALSAIEMANLF KSLRLS_Aq_DS03 (E95C-A101C, I82C-K131CG) (SEQ ID NO: 312)QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLCRGATPHFDYIASCVSKGLCDLSLELRKPITFGVITADTLEQAIERAGTCGHGNKGWEAALSAIEMANLF KSLR

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a lumazine synthase subunit including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to amino acid sequence set forth as any oneof SEQ ID NOs: 25-28 or 306-312.

c. DNA Starvation/Stationary Phase Protection Protein (DPS)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to asubunit of a DNA starvation/stationary phase protection protein (DPS)complex, such as a DPS subunit from Thermosynechococcus elongates,Kineococcuc radiotolerans, or Nostoc punctiforme, to construct aself-assembling DPS nanoparticle carrier including a DPS nanoparticlefused to a plurality of the heterologous carrier proteins. Non-limitingexamples of the sequence of DPS subunits that can be included in thefusion proteins of the self-assembling protein nanoparticle carrier areprovided as:

DNA starvation/stationary phase protection protein(Thermosynechococcus elongates) (SEQ ID NO: 29)SATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELAERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLA KGDGLVSDNA starvation/stationary phase protection protein(Kineococcuc radiotolerans) (SEQ ID NO: 30)TTIHDVQTTGLTQDAVTGFDASSRLNAGLQEVLVDLTALHLQGKQAHWNIVGENWRDLHLQLDTLVEAARGFSDDVAERMRAVGGVPDARPQTVAASRIGDVGPDEIDTRACVEAIVALVRHTVDTIRRVHDPIDAEDPASADLLHAITL ELEKQAWMIGSENRSPRRDNA starvation/stationary phase protection protein (Nostoc punctiforme)(SEQ ID NO: 31) SETQTLLRNFGNVYDNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLHEFFNEAYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQMVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLSHFLAKDSLTLGFVQAAQS

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a DPS subunit including an amino acid sequence atleast 80% (such as at least 85%, at least 90%, at least 95%, or at least97%) identical to amino acid sequence set forth as any one of SEQ IDNOs: 29-31.

d. Bacteriophage Q Beta Capsid Protein (qbeta)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to asubunit of a Bacteriophage Q Beta Capsid protein (qbeta) complex toconstruct a self-assembling qbeta nanoparticle carrier including a qbetananoparticle fused to a plurality of the heterologous carrier proteins.A non-limiting example of the sequence of a qbeta subunit that can beincluded in the fusion proteins of the self-assembling proteinnanoparticle carrier is provided as:

(SEQ ID NO: 32) AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANG A CDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a qbeta subunit including an amino acid sequence atleast 80% (such as at least 85%, at least 90%, at least 95%, or at least97%) identical to the amino acid sequence set forth as SEQ ID NO: 32.

e. Dihydrolipoyl Transacetylase Protein (e2p)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to asubunit of a dihydrolipoyl transacetylase protein (e2p) complex toconstruct a self-assembling e2p nanoparticle carrier including an e2pnanoparticle fused to a plurality of the heterologous carrier proteins.E2p nanoparticles are formed from 60 copies of the e2p subunit;structural information is deposited at the Protein Data Bank No. 1B5S.In the globular e2p nanoparticle, the N-terminus of the subunit issurface exposed and the C-terminus of the subunit is inside the globularnanoparticle. A non-limiting example of the sequence of an ep2 subunitthat can be included in the fusion proteins of the self-assemblingprotein nanoparticle carrier is provided as:

(SEQ ID NO: 33) AAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTAIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to an e2p subunit including an amino acid sequence atleast 80% (such as at least 85%, at least 90%, at least 95%, or at least97%) identical to the amino acid sequence set forth as SEQ ID NO: 33.

f. Phosphopantetheine Adenylyltransferase (6ccq)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to asubunit of a Phosphopantetheine Adenylyltransferase (6ccq) complex toconstruct a self-assembling 6ccq nanoparticle carrier including a 6ccqnanoparticle fused to a plurality of the heterologous carrier proteins.Phosphopantetheine Adenylyltransferase nanoparticles are formed from 6copies of the Phosphopantetheine Adenylyltransferase subunit; structuralinformation is deposited at the Protein Data Bank No. 6CCQ. Anon-limiting example of the sequence of a 6ccq subunit that can beincluded in the fusion proteins of the self-assembling proteinnanoparticle carrier is provided as:

(SEQ ID NO: 34) MQKRAIYPGTFDPITNGHIDIVTRATQMFDHVILAIAASPSKKPMFTLEERVALAQQATAHLGNVEVVGFSDLMANFARNQHATVLIRGLRAVADFEYEMQLAHMNRHLMPELESVFLMPSKEWSFISSSLVKEVARHQGDVTHFLPENV HQALMAKLAVD

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a 6ccq subunit including an amino acid sequence atleast 80% (such as at least 85%, at least 90%, at least 95%, or at least97%) identical to the amino acid sequence set forth as SEQ ID NO: 34.

g. Glutamate Synthase (1f52)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to asubunit of a Glutamate Synthase (1f52) protein complex to construct aself-assembling Glutamate Synthase nanoparticle carrier including aGlutamate Synthase nanoparticle fused to a plurality of the heterologouscarrier proteins. A non-limiting example of the sequence of a GlutamateSynthase subunit that can be included in the fusion proteins of theself-assembling protein nanoparticle carrier is provided as:

(SEQ ID NO: 35) EHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDR VRMTPHPVEFELYYSV

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a Glutamate Synthase subunit including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to the amino acid sequence set forth as SEQID NO: 35.

h. Calcium/Calmodulin Dependent Protein Kinase IIa (CaMKIIa), C-TerminalFragment (5U6Y)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to ac-terminal fragment of a Calcium/calmodulin dependent protein kinase IIa(CaMKIIa) protein to construct a self-assembling CaMKIIa nanoparticlecarrier including a nanoparticle based on the C-terminal fragment ofCaMKIIa fused to a plurality of the heterologous carrier proteins. TheCaMKIIa nanoparticle is formed from 12 copies of the c-terminal fragmentof CaMKIIa subunit; structural information is deposited at the ProteinData Bank No. 5U6Y. The N-terminus of the c-terminal fragment is surfaceexposed in the globular nanoparticle. Non-limiting examples of CaMKIIasequences that can be included in the fusion proteins of theself-assembling protein nanoparticle carrier are provided as:

(SEQ ID NO: 36) GGKSGGNKKSDGVKESSESTNTAIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVEGLDFHRFYFENLWSRNSKPVHTTILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSEETRVWHRRDGKWQI VHFHRSGA(SEQ ID NO: 37) GVKESSESTNTAIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVEGLDFHRFYFENLWSRNSKPVHTTILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSEETRVWHRRDGKWQIVHFHRSGA (SEQ ID NO: 38)STNTAIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVEGLDFHRFYFENLWSRNSKPVHTTILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSEETRVWHRRDGKWQIVHFHRSGA

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a Glutamate Synthase subunit including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to the amino acid sequence set forth as anyone of SEQ ID NOs: 36-38.

i. HIV Capsid Oligomerization Domain (HIV)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to a HIVcapsid oligomoization domain (HIV) to construct a self-assembling HIVcapsid oligomerization domain nanoparticle carrier including ananoparticle based on the HIV capsid oligomerization domain fused to aplurality of the heterologous carrier proteins. Non-limiting examples ofHIV capsid oligomerization domain sequences that can be included in thefusion proteins of the self-assembling protein nanoparticle carrier areprovided as:

(SEQ ID NO: 39) PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARV (SEQ ID NO: 40)PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKT ILKALGPGATLEEMMTA

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a HIV capsid oligomerization domain including an aminoacid sequence at least 80% (such as at least 85%, at least 90%, at least95%, or at least 97%) identical to the amino acid sequence set forth asany one of SEQ ID NOs: 39-40.

j. Hexamer

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to aHexamer subunit to construct a hexamer nanoparticle carrier including ananoparticle based on the hexamer sequence fused to a plurality of theheterologous carrier proteins. A non-limiting examples of a hexamersequence that can be included in the fusion proteins of theself-assembling protein nanoparticle carrier is provided as:

(SEQ ID NO: 41) PTLYNVSLVMSDTAGTCY

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a hexamer subunit including an amino acid sequence atleast 80% (such as at least 85%, at least 90%, at least 95%, or at least97%) identical to the amino acid sequence set forth as SEQ ID NO: 41.

k. T4 Fibritin Foldon Domain (Fd)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to a T4fibritin Foldon domain to construct a hexamer nanoparticle carrierincluding a nanoparticle based on the T4 fibritin Foldon domain sequencefused to a plurality of the heterologous carrier proteins. Anon-limiting examples of a T4 fibritin Foldon domain sequence that canbe included in the fusion proteins of the self-assembling proteinnanoparticle carrier is provided as:

(SEQ ID NO: 42) GYIPEAPRDGQAYVRKDGEWVLLSTFL

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a T4 fibritin Foldon domain including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to the amino acid sequence set forth as SEQID NO: 42.

l. Encapsulin

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to anencapsulin subunit to construct a self-assembling encapsulinnanoparticle carrier including an encapsulin nanoparticle fused to aplurality of the heterologous carrier proteins. Encapsulin nanoparticlesare formed from 60 copies of the encapsulin subunit.

The globular form of the encapsulin nanoparticle is made up of monomericsubunits. A non-limiting example of the sequence of an encapsulinsubunit is provided as:

(SEQ ID NO: 43) MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKF

Additional encapsulin subunits are provided with one or more cysteinesubstitutions to introduce non-native disulfide bond(s) that stabilizethe encapsulin nanoparticle formed from self-assembled subunits. In someembodiments, the non-native disulfide bond(s) are introduced withG53C-R94C, G53C-K96C, or K146C-A185C substitutions, or a combinationthereof. The residues numbering is with reference to the encapsulinsubunit set forth as SEQ ID NO: 43. Non-limiting examples include:

EN G53C-R94C (SEQ ID NO: 313)MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKFEN G53C-K96C (SEQ ID NO: 314)MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLErGcPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKFEN K146C-A185C (SEQ ID NO: 315)MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLgEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLErGkPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPcDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEcGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETF TFQVVNPEALILLKF

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to an encapsulin subunit including an amino acid sequenceat least 80% (such as at least 85%, at least 90%, at least 95%, or atleast 97%) identical to amino acid sequence set forth as SEQ ID NO: 43or 313-315.

Encapsulin proteins are a conserved family of bacterial proteins alsoknown as linocin-like proteins that form large protein assemblies thatfunction as a minimal compartment to package enzymes. The encapsulinassembly is made up of monomeric subunits, which are polypeptides havinga molecule weight of approximately 30 kDa. Following production, themonomeric subunits self-assemble into the globular encapsulin assemblyincluding 60, or in some cases, 180 monomeric subunits. Methods ofconstructing encapsulin nanoparticles are described, for example, inSutter et al. (Nature Struct. and Mol. Biol., 15:939-947, 2008, which isincorporated by reference herein in its entirety). In specific examples,the encapsulin polypeptide is bacterial encapsulin, such as Thermotogamaritime or Pyrococcus furiosus or Rhodococcus erythropolis orMyxococcus xanthus encapsulin.

m. Acinetobacter Phage AP205 (AP205)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to aAcinetobacter phage AP205 domain to construct a self-assembingnanoparticle carrier including a nanoparticle based on the Acinetobacterphage AP205 domain sequence fused to a plurality of the heterologouscarrier proteins. A non-limiting examples of an Acinetobacter phageAP205 domain sequence that can be included in the fusion proteins of theself-assembling protein nanoparticle carrier is provided as:

AP205 (SEQ ID NO: 316)MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTT

Additional Acinetobacter phage AP205 subunits are provided with one ormore cysteine substitutions to introduce non-native disulfide bond(s)that stabilize the Acinetobacter phage AP205nanoparticle formed fromself-assembled subunits. In some embodiments, the non-native disulfidebond(s) are introduced with T81C (which forms a disulfide with acysteine already present in AP205), S53C-H100C, or V82C-R80Csubstitutions, or a combination thereof. The residues numbering is withreference to the Acinetobacter phage AP205 subunit set forth as SEQ IDNO: 316. Non-limiting examples include:

AP205-T81C (SEQ ID NO: 317)MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIRcVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTT AP205 S53C-H100C (SEQ ID NO: 318)MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVcVYKRPAPKPEGCADACVIMPNENQSIRTVISGSAENLATLKAEWET$KRNVDTLFASGNAGLGFLDPTAAIVSSDTT AP205 V82C-R80C (SEQ ID NO: 319)MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADACVIMPNENQSIctcISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTT AP205 C65-C69GC (SEQ ID NO: 320)MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPEGCADAgCVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIVSSDTT

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a Acinetobacter phage AP205 subunit including an aminoacid sequence at least 80% (such as at least 85%, at least 90%, at least95%, or at least 97%) identical to the amino acid sequence set forth asSEQ ID NO: 316-320.

n. Hepatitis B Capsid Protein (HBV)

In some embodiments, any of the disclosed heterologous carrier proteins(such as an rTT, CRM197, or HiD carrier protein) can be linked to aHepatitis B capsid protein domain to construct a self-assemblingnanoparticle carrier including a nanoparticle based on the Hepatitis Bcapsid protein domain sequence fused to a plurality of the heterologouscarrier proteins. A non-limiting examples of an Hepatitis B capsidprotein domain sequence that can be included in the fusion proteins ofthe self-assembling protein nanoparticle carrier is provided as:

HBV (SEQ ID NO: 321) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

Additional Hepatitis B capsid protein subunits are provided with one ormore cysteine substitutions to introduce non-native disulfide bond(s)that stabilize the Hepatitis B capsid protein domain nanoparticle formedfrom self-assembled subunits. In some embodiments, the non-nativedisulfide bond(s) are introduced with P25C-R127C, E14C-A36C, D29C-R127C,F18C-A36C, or D29C-R127C substitutions, or a combination thereof. Theresidues numbering is with reference to the Hepatitis B capsid proteinsubunit set forth as SEQ ID NO: 321. Non-limiting examples include:

HBV P25C-R127C (SEQ ID NO: 322)MDIDPYKEFGATVELLSFLPSDFFcSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC HBV E14C-A36C (SEQ ID NO: 323)MDIDPYKEFGATVcLLSFLPSDFFPSVRDLLDTAScLYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC HBV D29C-R127C (SEQ ID NO: 324)MDIDPYKEFGATVELLSFLPSDFFPSVRcLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC HBV_DS01 (F18C-A36C) (SEQ ID NO: 325)MDIDPYKEFGATVELLSCLPSDFFPSVRDLLDTASCLYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC HBV_DS02 (D29C-R127C) (SEQ ID NO: 326)MDIDPYKEFGATVELLSFLPSDFFPSVRCLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWICTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC

In some embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise any of the disclosed heterologous carrierproteins fused to a Hepatitis B capsid subunit including an amino acidsequence at least 80% (such as at least 85%, at least 90%, at least 95%,or at least 97%) identical to the amino acid sequence set forth as anyone of SEQ ID NO: 321-326.

2. Heterologous Carrier Proteins

The heterologous carrier protein included in the fusion protein can beany carrier protein suitable for use as with a vaccine that is a singlepolypeptide chain of amino acids (as opposed to a protein complex).Examples of suitable heterologous carrier proteins are those that canincrease the immunogenicity of the conjugate and/or elicit antibodiesagainst the carrier which are diagnostically, analytically, and/ortherapeutically beneficial. Specific, non-limiting examples of suitablepolypeptide carriers include, but are not limited to, natural,semi-synthetic or synthetic polypeptides or proteins from bacteria orviruses. In one embodiment, bacterial products for use as carriersinclude bacterial toxins, such as those that are single polypeptidechains (or a fragment thereof) that mediate toxic effects, inflammatoryresponses, stress, shock, chronic sequelae, or mortality in asusceptible host. Specific, non-limiting examples of such bacterialtoxins include, but are not limited to: single polypeptide chains of B.anthracis PA (for example, as encoded by bases 143779 to 146073 ofGENBANK® Accession No. NC 007322); B. anthracis LF (for example, asencoded by the complement of bases 149357 to 151786 of GENBANK®Accession No. NC 007322); bacterial toxins and toxoids, such as tetanustoxin/toxoid (for example, as described in U.S. Pat. Nos. 5,601,826 and6,696,065); diphtheria toxin/toxoid (for example, as described in U.S.Pat. Nos. 4,709,017 and 6,696,065), such as tetanus toxin heavy chain Cfragment; P. aeruginosa exotoxin/toxoid (for example, as described inU.S. Pat. Nos. 4,428,931, 4,488,991 and 5,602,095); pertussistoxin/toxoid (for example, as described in U.S. Pat. Nos. 4,997,915,6,399,076 and 6,696,065); and C. perfringens exotoxin/toxoid (forexample, as described in U.S. Pat. Nos. 5,817,317 and 6,403,094) C.difficile toxin B or A, or analogs or mimetics of and combinations oftwo or more thereof. Viral proteins, such as hepatitis B surface antigen(for example, as described in U.S. Pat. Nos. 5,151,023 and 6,013,264)and core antigen (for example, as described in U.S. Pat. Nos. 4,547,367and 4,547,368) can also be used as carriers, as well as singlepolypeptide chains of proteins from higher organisms such as keyholelimpet hemocyanin (KLH), horseshoe crab hemocyanin, ConcholepasConcholepas Hemocyanin (CCH), Ovalbumin (OVA), edestin, mammalian serumalbumins (such as bovine serum albumin), and mammalian immunoglobulins.

In some embodiments, the heterologous carrier protein is selected fromone of: a Keyhole Limpet Hemocyanin (KLH) subunit, recombinant tetanustoxin heavy chain C fragment (rTT), diphtheria toxin variant CRM197, orH. influenzae protein D (HiD). CRM197 is a genetically detoxified formof diphtheria toxin; a single mutation at position 52, substitutingglutamic acid for glycine, causes the ADP-ribosyltransferase activity ofthe native diphtheria toxin to be lost. For description of exemplaryprotein carriers for vaccines, see Pichichero, Protein carriers ofconjugate vaccines: characteristics, development, and clinical trials,Hum Vaccin Immunother., 9: 2505-2523, 2013, which is incorporated byreference herein in its entirety).

In some embodiments, the heterologous carrier protein is an rTT protein,for example, comprising the amino acid sequence set forth as:

(SEQ ID NO: 44) MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

In some embodiments, the heterologous carrier protein is an rTT proteincomprising amino acid substitutions to remove one or more N-linkedglycosylation sites. It is believed that removal of the N-linkedglycosylation sites may improve accessibility of the protein surface forconjugation to the HIV-1 Env fusion peptide. Exemplary rTT proteinsequences with modifications to remove one or more N-linkedglycosylation sites are provided as:

(SEQ ID NO: 45) NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNS A VITYPDAQLVPGINGKAIHLVNNE A SEVIVHKAMDIEYNDMFN Q FTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDN QITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLK Q ITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 46)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

In some embodiments, the heterologous carrier protein is an rTT proteincomprising amino acid substitutions to remove one or more N-linkedglycosylation sites as well as to introduce lysine residues at surfaceexposed positions of the carrier. Increasing the number of lysineresidues in the heterologous carrier protein increases the number ofavailable sites for conjugation to the HIV-1 Env fusion peptides withmethods targeting the amino moiety of lysine, such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (Sulfo-SIAB) linkers. Exemplary rTT proteinsequences with modifications to remove one or more N-linkedglycosylation sites and/or to add lysine residues are provided as:

(SEQ ID NO: 47) NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 48)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 49)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 50)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 51)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 52)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 53)NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 54)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 55)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 56)NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 57)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 58)NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 59)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 60)NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 61)NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 62)NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND (SEQ ID NO: 63)NLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND

In some embodiments, the heterologous carrier protein is a fragment ofthe rTT protein, such as a fragment of rTT protein comprising,consisting essentially of, or consisting of the amino acid sequence setforth as:

(SEQ ID NO: 64) NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSIT

In some embodiments, the fusion protein can include an rTT sequence setforth as any one of SEQ ID NOs: 44-64, or an amino acid sequence atleast 90% identical thereto.

In some embodiments, the heterologous carrier protein is a HiD protein,for example, comprising the amino acid sequence set forth as:

(SEQ ID NO: 65) SNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK

In some embodiments, the fusion protein can include a HiD sequence setforth as SEQ ID NO: 65, or an amino acid sequence at least 90% identicalthereto.

In some embodiments, the heterologous carrier protein is a HiD proteincomprising amino acid substitutions to remove one or more N-linkedglycosylation sites and/or to introduce lysine residues at surfaceexposed positions of the carrier. It is believed that removal of theN-linked glycosylation sites may improve accessibility of the proteinsurface for conjugation to the HIV-1 Env fusion peptide.

In some embodiments, the heterologous carrier protein is a CRM197protein, for example, comprising the amino acid sequence set forth as:

(SEQ ID NO: 66) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

In some embodiments, the fusion protein can include a CRM197 sequenceset forth as SEQ ID NO: 66, or an amino acid sequence at least 90%identical thereto.

In some embodiments, the heterologous carrier protein is a CRM197protein comprising amino acid substitutions to remove one or moreN-linked glycosylation sites. It is believed that removal of theN-linked glycosylation sites may improve accessibility of the proteinsurface for conjugation to the HIV-1 Env fusion peptide. An exemplaryCRM197 protein sequence with modifications to remove one or moreN-linked glycosylation sites is provided as:

(SEQ ID NO: 67) GADDVVDSSKSFVMENF A SYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNK A VSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNK A KTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

In some embodiments, the fusion protein can include a CRM197 sequenceset forth as SEQ ID NOs: 67, or an amino acid sequence at least 90%identical thereto.

In some embodiments, the heterologous carrier protein is a Meningococcalouter membrane protein complex (OMPC) protein. An exemplary OMPC proteinsequence with modifications to remove one or more N-linked glycosylationsites is provided as:

(SEQ ID NO: 222) DFTIQDIRVEGLQRTEPSTVFNYLPVKVGDTYNDTHGSAIIKSLYATGFFDDVRVETADGQLLLTVIERPTIGSLNITGAKMLQNDAIKKNLESFGLAQSQYFNQATLNQAVAGLKEEYLGRGKLNIQITPKVTKLARNRVDIDITIDEGKSAKITDIEFEGNQVYSDRKLMRQMSLTEGGIWTWLTRSNQFNEQKFAQDMEKVTDFYQNNGYFDFRILDTDIQTNEDKTKQTIKITVHEGERFRWGKVSIEGDTNEVPKAELEKLLTMKPGKWYERQQMTAVLGEIQNRMGSAGYAYSEISVQPLPNAETKTVDFVLHIEPGRKIYVNEIHITGNNKTRDEVVRRELRQMESAPYDTSKLQRSKERVELLGYFDNVQFDAVPLAGTPDKVDLNMSLTERSTGSLDLSAGWVQDTGLVMSAGVSQDNLFGTGKSAALRASRSKTTLNGSLSFTDPYFTADGVSLGYDVYGKAFDPRKASTSIKQYKTTTAGAGIRMSVPVTEYDRVNFGLVAEHLTVNTYNKAPKHYADFIKKYGKTDGTDGSFKGWLYKGTVGWGRNKTDSALWPTRGYLTGVNAEIALPGSKLQYYSATHNQTWFFPLSKTFTLMFGGEVGIAGGYGKTKEIPFFENFYGGGLGSVRGYESGTLGPKVYDEYGEKISYGGNKKANVSAELLFPMPGAKDARTVRLSLFADAGSVWDGKTYDDNSSSATGGRVQNIYGAGNTHKSTFTNELRYSAGGAVTWLSPLGPMKFSYAYPLKKKPEDEI QRFQFQLGTTF

In some embodiments, the heterologous carrier protein is an OMPC proteincomprising amino acid substitutions to remove one or more N-linkedglycosylation sites and/or to introduce lysine residues at surfaceexposed positions of the carrier. It is believed that removal of theN-linked glycosylation sites may improve accessibility of the proteinsurface for conjugation to the HIV-1 Env fusion peptide.

In some embodiments, the heterologous carrier protein is anOuter-membrane lipoprotein carrier protein comprising amino acidsubstitutions to remove one or more N-linked glycosylation sites. It isbelieved that removal of the N-linked glycosylation sites may improveaccessibility of the protein surface for conjugation to the HIV-1 Envfusion peptide. An exemplary Outer-membrane lipoprotein carrier proteinsequence with modifications to remove one or more N-linked glycosylationsites is provided as:

(SEQ ID NO: 223) QAGAVDALKQFNNDADGISGSFTQTVQSKKKTQTAHGTFKILRPGLFKWEYTSPYKQTIVGDGQTVWLYDVDLAQVTKSSQDQAIGGSPAAILSNKTALESSYTLKEDGSSNGIDYVLATPKRNNAGYQYIRIGFKGGNLAAMQLKDSFGNQTSISFGGLNTNPQLSRGAFKFTPPKGVDVLSN

In some embodiments, the heterologous carrier protein is aOuter-membrane lipoprotein carrier protein comprising amino acidsubstitutions to remove one or more N-linked glycosylation sites and/orto introduce lysine residues at surface exposed positions of thecarrier. It is believed that removal of the N-linked glycosylation sitesmay improve accessibility of the protein surface for conjugation to theHIV-1 Env fusion peptide.

In some embodiments, the heterologous carrier protein is a Cholera ToxinB Subunit comprising amino acid substitutions to remove one or moreN-linked glycosylation sites. It is believed that removal of theN-linked glycosylation sites may improve accessibility of the proteinsurface for conjugation to the HIV-1 Env fusion peptide. An exemplaryCholera Toxin B Subunit sequence with modifications to remove one ormore N-linked glycosylation sites is provided as:

(SEQ ID NO: 224) NGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAIS MAN

In some embodiments, the heterologous carrier protein is a Cholera ToxinB Subunit comprising amino acid substitutions to remove one or moreN-linked glycosylation sites and/or to introduce lysine residues atsurface exposed positions of the carrier. It is believed that removal ofthe N-linked glycosylation sites may improve accessibility of theprotein surface for conjugation to the HIV-1 Env fusion peptide.

Any one of the above disclosed heterologous carrier proteins can befused to any one of the self-assembling protein nanoparticle subunits inthe fusion protein of the self-assembling protein nanoparticle carrier.

3. Linker

The heterologous carrier protein fused to the self-assembling proteinnanoparticle subunit can be direct linked (for example, the C-terminusof the heterologous carrier protein is linked to the N-terminus of theself-assembling protein nanoparticle subunit by a peptide bond), orindirectly linked by a peptide linker (for example, the C-terminus ofthe heterologous carrier protein is directly linked to the N-terminus ofa peptide linker by a peptide bond, and the C-terminus of the peptidelinker is directly linked to the N-terminus of the self-assemblingprotein nanoparticle subunit by a peptide bond). Any suitable linker canbe used to fuse the heterologous carrier protein and the self-assemblingprotein nanoparticle. In some embodiments, the linker comprises a camelIgG2a hinge (referred to as caIgG2a, EPKIPQPQPKPQPQPQPQPKPQPKPEPE, SEQID NO: 327). In some embodiments, the linker comprises a CD8 hingeregion, such as KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ IDNO: 328). In some embodiments, the linker comprises an antibody hingesequence, such as ggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsgg (SEQ IDNO: 329). In some embodiments, the linker comprises a flexible proteinsequence, such as a glycine serine linker sequence, for example,GGGGSGGGGS (SEQ ID NO: 330).

The linker fusing the carrier protein and the self-assemblingnanoparticle subunit can be any suitable length; in some embodiments,the linker is from 10-00 amino acids in length, such as from 10-50 aminoacids in length.

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 44 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 45 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 46 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 47 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 48 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 49 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 50 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 51 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 52 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 53 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 54 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 55 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 56 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 57 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 58 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 59 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 60 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 61 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 62 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 63 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a rTTcarrier protein such as SEQ ID NO: 64 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a HiDcarrier protein such as SEQ ID NO: 65 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of aCRM197 carrier protein such as SEQ ID NO: 66 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of aCRM197 carrier protein such as SEQ ID NO: 67 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of a OMPCcarrier protein such as SEQ ID NO: 222 linked to any one of theself-assembling protein nanoparticle subunits provided herein by apeptide linker, such as a caIgG2a linker (e.g., SEQ ID NO: 327), a CD8linker (e.g., SEQ ID NO: 328), an antibody hinge linker (e.g., SEQ IDNO: 329), or a flexible linker such as a glycine-serine linker (e.g.,SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of aOuter-membrane lipoprotein carrier protein such as SEQ ID NO: 223 linkedto any one of the self-assembling protein nanoparticle subunits providedherein by a peptide linker, such as a caIgG2a linker (e.g., SEQ ID NO:327), a CD8 linker (e.g., SEQ ID NO: 328), an antibody hinge linker(e.g., SEQ ID NO: 329), or a flexible linker such as a glycine-serinelinker (e.g., SEQ ID NO: 330).

In some embodiments, the fusion protein comprises or consists of aCholera Toxin B Subunit carrier protein such as SEQ ID NO: 224 linked toany one of the self-assembling protein nanoparticle subunits providedherein by a peptide linker, such as a caIgG2a linker (e.g., SEQ ID NO:327), a CD8 linker (e.g., SEQ ID NO: 328), an antibody hinge linker(e.g., SEQ ID NO: 329), or a flexible linker such as a glycine-serinelinker (e.g., SEQ ID NO: 330).

4. Heterologous T-Cell Helper Epitope

In some embodiments, the fusion protein further comprises a heterologousT-cell helper epitope sequence. It is believed that the presence of theheterologous T-cell helper epitope sequence on the self-assemblingprotein nanoparticle carrier will improve the immune response elicitedby an immunogenic conjugate containing the carrier conjugated to HIV-1Env fusion peptides as disclosed herein. Any suitable heterologousT-cell helper epitope sequence can be included on the fusion protein. Insome embodiments, the amino acid sequence of the T-cell helper epitopeis the sequence of a pan DR epitope (PADRE), such as AKFVAAWTLKAAA (SEQID NO: 221). In some embodiments, the amino acid sequence of the T-cellhelper epitope is the sequence of a P2 epitope, such as QYIKANSKFIGITEL(SEQ ID NO: 68). In some embodiments, the amino acid sequence of theT-cell helper epitope is the sequence of a TpD epitope, such asILMQYIKANSKFIGKVSVRQSIALSSLMVAQ (SEQ ID NO: 69). In some embodiments,the amino acid sequence of the T-cell helper epitope is the sequence ofan HIV-1 Env epitope, such as HIV-1 Env residues 31-45 according to theHXB2 numbering system, for example, AENLWVTVYYGVPVW (SEQ ID NO: 70) orTEKLWVTVYYGVPVW (SEQ ID NO: 71). In some embodiemnts, the amino acidsequence of the T-cell helper epitope is selected from any one of:

(a) SEQ ID NO: 67;

(b) SEQ ID NO: 68;

(c) SEQ ID NO: 69;

(d) the sequence of HIV-1 Env residues 31-45 according to the HXB2numbering system (Env31-45 epitope); or

(e) a combination of any one of (a) and (b); (a) and (c); (a) and (d);(b) and (c); (b) and (d); (c) and (d); (a), (b), and (c); (a), (b), and(d); (a), (c), and (d); (b), (c), and (d); or (a), (b), (c), and (d).

In some embodiments, the amino acid sequence of the T-cell helperepitope is the sequence of a p458m epitope, such as NEDQKIGIEIIKRALKI(SEQ ID NO: 225). In some embodiments, the amino acid sequence of theT-cell helper epitope is the sequence of a P30 epitope, such asFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 226). In some embodiments, the aminoacid sequence of the T-cell helper epitope is the sequence of adiphtheria toxin epitope, such as PVFAGANYAAWAVNVAQVI (DTD271-290, SEQID NO: 227), HHNTEEIVAQSIALSSLMV (DTD321-340, SEQ ID NO: 228),QSIALSSLMVAQAIPLVGEL (DTD331-350, SEQ ID NO: 229), VDIGFAAYNFVESIINLFQV(DTD351-370, SEQ ID NO: 230), QGESGHDIKITAENTPLPIA (DTD411-430, SEQ IDNO: 231), or GVLLPTIPGKLDVNKSKTHI (DTD431-450, SEQ ID NO: 232). In someembodiments, the amino acid sequence of the T-cell helper epitope is thesequence of a tetanus toxin epitope, such as NSVDDALINSTKIYSYFPSV (TT580-599, SEQ ID NO: 233), QYIKANSKFIGITEL (TT 830-844, SEQ ID NO: 234),PGINGKAIHLVNNESSE (TT, 916-932, SEQ ID NO: 235), FNNFTVSFWLRVPKVSASHLE(TT, 947-967, SEQ ID NO: 236). In some embodiments, the amino acidsequence of the T-cell helper epitope is the sequence of an HIV-1 Envepitope, such as TQLFNSTWFNSTWST (HIV-1 Env 388-402, SEQ ID NO: 237),EQIWNHTTWMEWDRE (HIV-1 Env 620-634, SEQ ID NO: 238),IRGQIRCSSNITGLLLTRDGGNNAAA (HIV_env_DRBO101_1, SEQ ID NO: 239),QCTHGIRPVVSTQLLLNGSLAEE (HIV_env_DRBO101_2, SEQ ID NO: 240),NDNTSYRLISCNTSVITQACPKV (HIV_env_DRBO101_3, SEQ ID NO: 241),SENFTNNAKIIIVQLNESVVINC (HIV_env_DRBO101_5, SEQ ID NO: 242),EVVIRSENFTNNAKTIIVQLNES (HIV_env_DRBO101_7, SEQ ID NO: 243),TVQCTHGIRPVVSTQLLLNGSLA (HIV_env_DRBO101_11, SEQ ID NO: 244), orESVVINCTRPNNNTRRSIHIGPG (HIV_env_DRBO101_14, SEQ ID NO: 245).

The heterologous T-cell helper epitope can be located at any suitablesection of the fusion protein, including (but not limited to) theN-terminus, the C-terminus, and between the heterologous carrier proteinand the self-assembling protein nanoparticle subunit. In someembodiments, the heterologous T-cell helper epitope is separated fromthe carrier protein and/or the self-assembling protein nanoparticlesubunit in the fusion protein by one or more peptide linkers.

5. Targeting Moiety

In some embodiments, the immunogenic conjugate further includes a moietythat targets the immune system in a subject to enhance the immuneresponse to the HIV-1 Env fusion peptide on the immunogenic conjugate.The moiety can be, for example, a moiety that binds to components of theimmune system in the subject, such as a pattern recognition receptor, adendritic cell, or to antigens located in B-cell developmental regionsof the immune system, such as germinal centers.

In some embodiments, the fusion protein is linked to a moiety thatspecifically binds to a pattern recognition receptor agonist, such as atoll-like receptor (TLR) agonist, a Stimulator of Interferon Genes(STING) agonist, a C-type lectin receptor (CLR) agonist, a RIG-I-likereceptor (RLR) agonist, or a NOD-like receptor (NLR) agonist.

In several embodiments, the moiety can be a pattern recognition receptoragonist. Non-limiting examples of pattern recognition receptor agonistsinclude TLR-1/2/6 agonists (e.g., lipopeptides and glycolipids, such asPam2cys or Pam3cys lipopeptides); TLR-3 agonists (e.g., dsRNA, such asPolyI:C, and nucleotide base analogs); TLR-4 agonist (e.g.,lipopolysaccharide (LPS) derivatives and small molecule analogs ofpyrimidoindole); TLR5 agonists (e.g., Flagellin); TLR-7/8 agonists(e.g., ssRNA and nucleotide base analogs, including derivatives ofimidazoquinolines, hydroxy-adenine, benzonapthyridine and loxoribine);and TLR-9 agonists (e.g., unmethylated CpG); Stimulator of InterferonGenes (STING) agonists (e.g., cyclic dinucleotides, such as cyclicdiadenylate monophosphate); C-type lectin receptor (CLR) agonists (suchas various mono, di, tri and polymeric sugars that can be linear orbranched, e.g., mannose, Lewis-X tri-saccharides, etc.); RIG-I-likereceptor (RLR) agonists; and NOD-like receptor (NLR) agonists (such aspeptidogylcans and structural motifs from bacteria, e.g.,meso-diaminopimelic acid and muramyl dipeptide); and combinationsthereof. In several embodiments, the pattern recognition receptoragonist can be a TLR agonist, such as an imidazoquinoline-based TLR-7/8agonist. For example, the adjuvant can be Imiquimod (R837) or Resiquimod(R848), which are approved by the FDA for human use.

In several embodiments, the moiety can be a TLR-7 agonist, a TLR-8agonist and/or a TLR-7/8 agonist. Numerous such agonists are known,including many different imidazoquinoline compounds. Imidazoquinolinesare synthetic immunomodulatory drugs that act by binding Toll-likereceptors 7 and 8 (TLR-7/TLR-8) on antigen presenting cells (e.g.,dendritic cells), structurally mimicking these receptors' naturalligand, viral single-stranded RNA. Imidazoquinolines are heterocycliccompounds comprising a fused quinoline-imidazole skeleton. Derivatives,salts (including hydrates, solvates, and N-oxides), and prodrugs thereofalso are contemplated by the present disclosure. Particularimidazoquinoline compounds are known in the art, see for example, U.S.Pat. Nos. 6,518,265; and 4,689,338. In some non-limiting embodiments,the imidazoquinoline compound is not imiquimod and/or is not resiquimod.

The moiety that targets the immune system in a subject can be linked tothe immunogenic conjugate by any suitable means.

In some embodiments, the fusion protein includes the sequence offlagellin subunit.

In some embodiments, the fusion protein of the self-assemblingprotein-nanoparticle carrier includes a streptavidin sequence, and themoiety that targets the immune system in a subject is biotinylated, forexample, a biotinylated pattern recognition receptor agonist, such as abiotinylated TLR agonist, a biotinylated STING agonist, a biotinylatedCLR agonist, a biotinylated RLR agonist, or a biotinylated NLR agonist.The biotinylated moiety can be linked to the self-assemblingprotein-nanoparticle carrier.

In some embodiments, the moiety that targets the immune system in thesubject is conjugated to the self-assembling protein nanoparticlecarrier using the same conjugate method as that used to conjugate theHIV-1 Env fusion peptide to the self-assembling protein nanoparticlecarrier. In some such embodiments, conjugation of the moiety thattargets the immune system and the HIV-1 fusion peptide to theself-assembling protein nanoparticle carrier can be completed in thesame reaction. For example, both the moiety that targets the immunesystem in the subject and the HIV-1 Env fusion peptide can be linked toa cysteine residue for conjugation to the self-assembling proteinnanoparticle carrier as described herein. In some embodiments, the HIV-1Env fusion peptide linked to a cysteine residue is mixed with a smallamount of TLR-7 or 8 agonist modified to include a reactive —SH group,so that both the HIV-1 Env fusion peptide and the TLR7/8-agonist areconjugated via a single reaction to a bifunctional crosslinker-activatedself-assembled protein nanoparticle carrier.

6. Exemplary Fusion Protein Embodiments

In several embodiments, the fusion proteins of the self-assemblingprotein nanoparticle carrier comprise, consist essentially of, orconsist of the amino acid sequence of any one of fusion proteins listedin the following table (showing SEQ ID NOs: 72-219, 246-257, and331-397), or an amino acid sequence at least 90% (such as at least 95%)identical to any one of SEQ ID NOs: 72-219, 246-257, or 331-397 thatself-assembles into a protein nanoparticle under suitable conditions. Insome embodiments, the fusion proteins of the self-assembling proteinnanoparticle carrier comprise, consist essentially of, or consist of theamino acid sequence set forth as any one of SEQ ID NOs: 73, 76, 79, 100,101, 109, 116, 167, 172, 180, 197, or 211, or an amino acid sequence atleast 90% (such as at least 95%) identical to any one of SEQ ID NOs: 73,76, 79, 100, 101, 109, 116, 167, 172, 180, 197, or 211 thatself-assembles into a protein nanoparticle under suitable conditions.

TABLE 1Exemplary sequences of fusion proteins containing a protein nanoparticle subunit fusedto a heterologous carrier protein and optionally a heterologous T-cell helper epitope.SEQ ID NO Name Sequence Lumazine Synthase 72 LS-20-CRMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRggksggnkksdgvkessesgGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS 73 LS-20-rTTQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRggksggnkksdgvkessesgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDE GWTND 74LS-20-HID QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRggksggnkksdgvkessesgSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK 75 LS-PADRE-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA Env31-CRMGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRSLVRAKFVAAWTLKAAAGSLVRAENLWVTVYYGVPVWslvrgGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS 76 LS-PADRE-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA Env31-rTTGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRSLVRSLVRAKFVAAWTLKAAAGSLVRAENLWVTVYYGVPVWslvrgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 77 LS-PADRE-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA Env31-HIDGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRSLVRSLVRAKFVAAWTLKAAAGSLVRAENLWVTVYYGVPVWslvrgSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIK 78 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggsgggsgggsMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR 79 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAA rTT_deglyGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGGSGGGQMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 80 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 10f-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsaeaaakeaaakaggggsggggsggggsggggsggggsggggsggggMQIYEGKLTAEGLREGIVASRENHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 81 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL r8-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDaeaaakeaaakeaaakeaaakaleaeaaakeaaakeaaakeaaakaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 82 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 12ap-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsapapapapapapapapapapapapasgMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 83 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 10pa-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDapapapapapapapapapapMQIYEGKLTAEGLRFGIVASRENHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMAN LFKSLR 84rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 2rf-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDaeaaakeaaakagsgsgsMQIYEGKLTAEGLREGIVASRENHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLF KSLR 85rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 3f-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSL R 86rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 5gs-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgsgsgsgsasgMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 87rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 3pa-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDapapapggggsMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 88 LS-r8-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA rTT_deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRaeaaakeaaakeaaakeaaakaleasaaakeaaakeaaakeaaakaMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 89 LS-8f-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA rTT_deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRggggsggggsggggsggggsggggsaeaaakeaaakaggggsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 90 LS-r6-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA rTT_deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRaeaaakeaaakeaaakaleasaaakeaaakeaaakaMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 91 LS-12ap-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA rTT_deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRgsapapapapapapapapapapapapasgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 92 LS-10pa-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA rTT_deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRapapapapapapapapapapMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTD EGWTND 93LS-f2r- MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVArTT_degly AGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgsgaeaaakeaaakaMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGW TND 94LS-3f- MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVArTT_degly AGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRggggsggggsggggsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTN D 95LS-5gs- MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVArTT_degly AGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgsgsgsgsasgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 96 LS-3pa-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA rTT_deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRggggspapapasgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 97 rTT-P2-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHL LS-PadreVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDsgsaGKKGSSQYIKANSKFIGITELMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRAKFVAAWTLKAAA 98 rTT-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHL linker-VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI LS-PadreGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRAKFVAAWTLK AAA 99rTT- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLalphaLinker-VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI LSGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDsgsaKALEAQKQK MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR 100 LS-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAalphaLinker-AGELARKENISAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQA rTTIERAGTKHGNKGWEAALSA/EMANLFKSLRsgsaKALEAQKQKMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 101rTT-LS- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLPADRE- VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSISaTyflagellin-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN CC-GVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSI YWTTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarke

i

aviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrGGKSGGNKKSDGVNLTDLGLTQSNIQKLDIDITEGDNAGVQITLTNDQALVKVGNFQTQGSVRDIENLRQTIEAQISDLDSQSNTSNASQVALERVRQLNNNIENLAGETTQAISIGDNANRSAQTLGKINATFRNAIAQGAADDKASNIRLGSSLREIATGLASQSKNLNNQTLLSLSNTNIVQAGSGSARLLSLVNQPVQNAQALVSTGAQQLIQARSMNSVETAYDSDEIRSRASTLNNVTNGLNTIASNFRNQVAGLDSRLTDVQALAADIK QLPNE 102rTT-LS- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLPADRE- VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSISaTyflagellin-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN CC-GVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSI YW-deglyTTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarke

i

aviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrGGKSGGNKKSDGVNLaDLGLTQSNIQKLDIDITEGDNAGVQITLTNDQALVKVGNFQTQGSVRDIENLRQTIEAQISDLDSQSNTSNAaQVALERVRQLNNNIENLAGETTQAISIGDNANRaAQTLGKINAaFRNAIAQGAADDKASNIRLGSSLREIATGLASQSKNLNNQaLLSLSNTNIVQAGSGSARLLSLVNQPVQNAQALVSTGAQQLIQARSMNSVETAYDSDEIRSRASTLNNVaNGLNTIASNFRNQVAGLDSRLTDVQALAADIK QLPNE 103revTT-LS- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLPADRE- VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSISaTyflagellin-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN CC-GVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSI YWTTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvypgsweipvaagelarke

i

aviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrGGKSGGNKKSDGVENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRGSGSAAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLG LDTLN 104revTT-LS- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLPADRE- VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSISaTyflagellin-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN CC-GVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSI YW-deglyTTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDgsgmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarke

i

aviaigvli rgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrGGKSGGNKKSDGVENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLaSARSRIEDSDYATEVSNMaRAQILQQAGTSVLAQANQVPQNVLSLLRGSGSAAQVINTNSLSLLTQNNLNKaQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSaNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQ TLGLDTLN105 SaTyflagellin-NLTDLGLTQSNIQKLDIDITEGDNAGVQITLTNDQALVKVGNFQTQGSVRDIENLRQTIEAQ LS-ISDLDSQSNTSNASQVALERVRQLNNNIENLAGETTQAISIGDNANRSAQTLGKINATFRNA PADRE-IAQGAADDKASNIRLGSSLREIATGLASQSKNLNNQTLLSLSNTNIVQAGSGSARLLSLVNQ rTT-His-PVQNAQALVSTGAQQLIQARSMNSVETAYDSDEIRSRASTLNNVTNGLNTIASNFRNQVAGL CC-YWDSRLTDVQALAADIKQLPNEGGKSGGNKKSDGVmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarkeNiSaviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTS YLSIT 106SaTyflagellin-NLaDLGLTQSNIQKLDIDITEGDNAGVQITLTNDQALVKVGNFQTQGSVRDIENLRQTIEAQ LS-ISDLDSQSNTSNAaQVALERVRQLNNNIENLAGETTQAISIGDNANRaAQTLGKINAaFRNA PADRE-IAQGAADDKASNIRLGSSLREIATGLASQSKNLNNQaLLSLSNTNIVQAGSGSARLLSLVNQ rTT-His-PVQNAQALVSTGAQQLIQARSMNSVETAYDSDEIRSRASTLNNVaNGLNTIASNFRNQVAGL CC-YW-DSRLTDVQALAADIKQLPNEGGKSGGNKKSDGVgsgmqiyegkltaeglrfgivasrfnhal deglyvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarkeNiSaviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKL YTSYLSIT107 revSaTyflagellin-ENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMS LS-PADRE-RAQILQQAGTSVLAQANQVPQNVLSLLRGSGSAAQVINTNSLSLLTQNNLNKSQSALGTAIE rTT-His-RLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRV CC-YWRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNGGKSGGNKKSDGVmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarkeNiSaviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgradlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTS YLSIT 108revSaTyflagellin-ENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLaSARSRIEDSDYATEVSNMa LS-PADRE-RAQILQQAGTSVLAQANQVPQNVLSLLRGSGSAAQVINTNSLSLLTQNNLNKaQSALGTAIE rTT-His-RLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRV CC-YW-RELAVQSANSaNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGE deglyTIDIDLKQINSQTLGLDTLNGGKSGGNKKSDGVgsgmqiyegkltaeglrfgivasrfnhalvdrlvegaidaivrhggreeditlvrvpgsweipvaagelarkeNiSaviaigvlirgLEVLFQGPGAKFVAAWTLKAAAGDEVDatphfdyiasevskgladlslelrkpitfgvitadtleqaieragtkhgnkgweaalsaiemanlfkslrggTTMLATRNFSGGKSGGNKKSDGVKESSESTNTTIEDEDMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSIT 246 LS-rTT-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA deglyAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgsgsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 247 LS-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA CRM197-AGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQA deglyIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgsgsDYKDDDDKgsgGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS 248 LS-MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVA 2xCRM197-AGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQA deglyIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgDYKDDDDKgsgGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgggsgggsGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSL FFEIKS249 LS-HIDMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgDYKDDDDKgsgsnmantqmksdkiiiahrgasgylpehtleskalafaqqadyleqdlamtkdgrlvvihdhfldgltdvakkfphrhrkdgryyvidftlkeiqslemtenfetkdgkqaqvypnrfplwkshfrihtfedeiefiqglekstgkkvgiypeikapwfhhqngkdiaaetlkvlkkygydkktdmvylqtfdfnelkriktellpqmgmdlklvqliaytdwketqekdpkgywvnynydwmfkpgamaevvkyadgvgpgwymlvnkeeskpdnivytplvkelaqynvevhpytvrkdalpefftdvnqmydallnksgatgvftdfpdtgveflkgik 250 TThc-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL degly-LSVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgdykddddkgsgMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLRgsgDYKDDDDKgsg 251 HID-LSSnmantqmksdkiiiahrgasgylpehtleskalafaqqadyleqdlamtkdgrlvvihdhfldgltdvakkfphrhrkdgryyvidftlkeigslemtenfetkdgkqaqvypnrfplwkshfrihtfedeiefiqglekstgkkvgiypeikapwfhhqngkdiaaetlkvlkkygydkktdmvylqtfdfnelkriktellpqmgmdlklvqliaytdwketqekdpkgywvnynydwmfkpgamaevvkyadgvgpgwymlvnkeeskpdnivytplvkelaqynvevhpytvrkdalpefftdvnqmydallnksgatgvftdfpdtgveflkgikgsgdykddddkgsgMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 252 CRM197-GADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA degly-LSAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgsgdykddddkgsgMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMANLFKSLR 253 2xCRM197-GADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA degly-LSAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgggsgggsGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgsgdykddddkgsgMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGCIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKCWEAALSAIEMAN LFKSLR331 LS K7C-QIYEGcLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVcHGGREEDITLVRVPGSWEIPVAA R40CGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI caIgG2a-ERAGTKHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEPE rTTgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 332 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131C-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI caIgG2a-ERAGTcHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEPE rTTgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 333 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131CGGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI caIgG2a-ERAGTcgHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEP rTTEgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 334 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131GCGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI caIgG2a-ERAGTgcHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEP rTTEgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 335 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121CG-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcgEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEP caIgG2a-EgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGK rTTAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 336 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121GC-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTgcEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEP caIgG2a-EgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGK rTTAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 337 LS K7C-QIYEGcLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVcHGGREEDITLVRVPGSWEIPVAA R40CGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI CD8v1-rTTERAGTKHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAtRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 338 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131C-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI CD8v1-rTTERAGTcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAtRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 339 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131CGGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI CD8v1-rTTERAGTcgHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAtRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 340 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131GCGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI CD8v1-rTTERAGTgcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAtRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 341 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121CG-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcgEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAt CD8v1-rTTRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 342 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121GC-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTgcEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAt CD8v1-rTTRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 343 LS K7C-QIYEGcLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVcHGGREEDITLVRVPGSWEIPVAA R40C C08-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI rTTERAGTKHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 344 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131C-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI CD8-rTTERAGTcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 345 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131CGGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI CD8-rTTERAGTcgHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 346 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131GCGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI CD8-rTTERAGTgcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 347 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121CG-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcgEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAC CD8-rTTRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 348 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121GC-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTgcEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAC CD8-rTTRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 349 LS K7C-QIYEGcLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVcHGGREEDITLVRVPGSWEIPVAA R40CGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI hinge-rTTERAGTKHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 350 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131CGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI hinge-rTTERAGTcHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 351 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131CGGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI hinge-rTTERAGTcgHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 352 LS-L121C-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA K131GCGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcEQAI hinge-rTTERAGTgcHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 353 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121CG-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTcgEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTH hinge-rTTTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 354 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA L121GC-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTgcEQA K131CIERAGTcHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTH hinge-rTTTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 355 LS-15-TT

ggggsggggsggggsMKNLDCWVDNEEDIDVIL KKSTILNLDINNDIISDISGFNSSVITYPDAQLV

VIVHKAMDIEY NDM

QYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 356LS-25-TT

ggksggnkksdgvkessesgggsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLV

NV IVHKAMDIEYNDM

QYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 357 LS-30-TT

ggggsggksggnkksdgvkessesgggsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLV

VIVHKAMDIEYNDM

QYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 358 LS-35-TT

ggksggnkksdgvkessesgggsggggggsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLV

VIVHKAMDIEYNDM

QYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 359 LS-20-

env31-TT

ggksggnkksdgvkSLVR

essesgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQL V

VIVHKAMDIEYND

QYGTNEYSI ISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 360 LS-20-

PADRE-TT

ggksggnkksdgvkeSLVR

essesgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQL V

VIVHKAMDIEYNDM

QYGTNEYSI ISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 361 LS-hinge-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA rTTGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR

KNLDCWVDN EEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVP TDEGWTND362 LS- QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAhinge2- GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIrTT ERAGTKHGNKGWEAALSAIEMANLFKSLR

KNLDCWVDNEED (removeIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKA the cysMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNL fromIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLG hinge)AIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDE GWTND 363LS- QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAhinge2.1- GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIrTT ERAGTKHGNKGWEAALSAIEMANLFKSLRggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 364 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA hinge3-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI rTTERAGTKHGNKGWEAALSAIEMANLFKSLRggEPKSTDKTHTSPPSPAPELLggKNLDCWVDN (mutateEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIV th cysHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKG to Thr inNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEIT hinge)GLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVP TDEGWTND365 LS-ext1-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA rTTGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI (extendERAGTKHGNKGWEAALSAIEMANLFKSLR

KNLDCWV the NDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEV terminalIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSL of rTT)KGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 366 LS-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA caIgG2a-GELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAI rTTERAGTKHGNKGWEAALSAIEMANLFKSLRgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEPEgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 367 LS-CD8-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA rTTGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEAtRPAAGGAVHTRGgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 368 LS-CD8v2-QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAA rTTGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRgsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND Ferritin 109 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 1NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsggASISEKMVEALNRQINAEIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSRRGGRVKLYAVEEPPSEWDSPLAAFEHVYEHEVNVVKRIHELVEMAMQEKDFATYNFLQWYVAEQVEEEASALDIVEKLRLIGEDKRALLFLDKELSLRQFTPPAEEEK 110 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 2NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsgggsggsgASISEKMVEALNRQINAEIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSRRGGRVKLYAVEEPPSEWDSPLAAFEHVYEHEVNVVKRIHELVEMAMQEKDFATYNFLQWYVAEQVEEEASALDIVEKLRLIGEDKRALLFLDKELSLRQFTPPAEEEK 111 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 3NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggdgggdgggdggdgASISEKMVEALNRQINAEIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSRRGGRVKLYAVEEPPSEWDSPLAAFEHVYEHEVNVVKRIHELVEMAMQEKDFATYNFLQWYVAEQVEEEASALDIVEKLRLIGEDKRALLFLDKELSLRQFTPPAEEEK 112 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 4NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsggMLSKDIIKLLNEQVNKEMDSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGI AKSRKS113 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 5NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsgggsggsgMLSKDIIKLLNEQVNKEMDSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 114 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 6NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggdgggdgggdggdgMLSKDIIKLLNEQVNKEMDSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 115 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 116rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL ln15-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGggsggSGGgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 117 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL ln25-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGggsggSGGggSGGggSGGgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 118 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL ln35-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGggsggSGGggSGGggSGGggSGGggSGGgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 119 rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K5A-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 120rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL K5B-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 121rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL K5C-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 122rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL K5D-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 123rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K10A-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 124rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL K10B-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 125rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K10C-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 126rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL K10D-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 127rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K15A-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 128rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL K15B-FerVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRKSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 129rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K15C-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 130rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K15D-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRKSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 131rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K20-FerVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRKSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK S 132rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K20-VNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSI ln15-FerGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGggsggSGGgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 133 rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K20-VNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSI ln25-FerGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGggsggSGGggSGGggSGGgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 134 rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL K20-VNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSI ln35-FerGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggSGGggsggSGGggSGGggSGGggSGGggSGGgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 135 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL r8-FerrVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggaeaaakeaaakeaaakeaaakaleaeaaakeaaakeaaakeaaakaDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 136 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 12pa-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI FerrGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsapapapapapapapapapapapapaDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 137 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL r3-FerrVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggaeaaakeaaakeaaakaDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 138 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 3f-FerrVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 139 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 2rf-FerrVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsaeaaakeaaakaDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYV KGIAKSRKS140 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 5ga-FerrVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgsgsgsgsasgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVK GIAKSRKS141 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 1rf-FerrVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDpapapasgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKS RKS 142Ferr_deltaCT1-DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV linker-PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQH rTTEEEVLFKDILDKIELIG KALEAQKQKMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 143 Ferr_deltaCT2-DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV linker-PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQH rTTEEEVLFKDILDKIELIGNEN KALEAQKQKMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 144 rTT-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHL linker-VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI FerrGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDKALEAQKQKSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGI AKSRKS145 rTT- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHL2xlinker- VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIFerr GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDKALEAQKQKKALEAQKQKSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 146 Ferr_deltaCT1-DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV linker-PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQH CRMEEEVLFKDILDKIELIG KALEAQKQKGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFE IKS 147Ferr_deltaCT2-DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNV linker-PVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQH CRMEEEVLFKDILDKIELIGNEN KALEAQKQKGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSL FFEIKS148 CRM- GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAlinker- AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIFerr KRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSKALEAQKQKSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 149 CRM-GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA 2xlinker-AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFI FerrKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSKALEAQKQKKALEAQKQKSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS 150 rTT-ferrNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 1NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGaDWYFVPTDEGWTNDggsgggsggASISEKMVEALNRQINAEIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQEELMHAMKMFDFVSRRGGRVKLYAVEEPPSEWDSPLAAFEHVYEHEVNVVKRIHELVEMAMQEKDFATYNFLQWYVAEQVEEEASALDIVEKLRLIGEDKRALLFLDKELSLRQFTPPAEEEK DNA starvation/stationary phase protection protein (DPS) 151dps(te)- SATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELArTT 1 ERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLAKGDGLVSggsgggsggKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 152 dps(te)-SATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELA rTT 2ERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLAKGDGLVSggsgggsgggsggsgKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGW TND 153dps(te)- SATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELArTT 3 ERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLAKGDGLVSggdsggdgggdggdgKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGW TND 154rTT- NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNdps(te) 1 NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsggSATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELAERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLAKGDGLVS 155 rTT-NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN dps(te) 2NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsggsggSATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELAERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLAKGDGLVS 156 rTT-NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN dps(te) 3NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggdggdggSATTTLKEQVLTTLKREQANAVVMYLNYKKYHWLTYGPLFRDLHLLFEEQGSEVFAMIDELAERSLMLDGQPVADPADYLKVATVTPSSGQLTVKQMIEEAIANHELIITEMHQDAEIATEAGDIGTADLYTRLVQTHQKHRWFLKEFLAKGDGLVS 157 dps(kr)-TTIHDVQTTGLTQDAVTGFDASSRLNAGLQEVLVDLTALHLQGKQAHWNIVGENWRDLHLQL rTT 1DTLVEAARGFSDDVAERMRAVGGVPDARPQTVAASRIGDVGPDEIDTRACVEAIVALVRHTVDTIRRVHDPIDAEDPASADLLHAITLELEKQAWMIGSENRSPRRggsggKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEG WTND 158dps(kr)- TTIHDVQTTGLTQDAVTGFDASSRLNAGLQEVLVDLTALHLQGKQAHWNIVGENWRDLHLQLrTT 2 DTLVEAARGFSDDVAERMRAVGGVPDARPQTVAASRIGDVGPDEIDTRACVEAIVALVRHTVDTIRRVHDPIDAEDPASADLLHAITLELEKQAWMIGSENRSPRRggsgggsggKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVP TDEGWTND159 dps(kr)-TTIHDVQTTGLTQDAVTGFDASSRLNAGLQEVLVDLTALHLQGKQAHWNIVGENWRDLHLQL rTT 3DTLVEAARGFSDDVAERMRAVGGVPDARPQTVAASRIGDVGPDEIDTRACVEAIVALVRHTVDTIRRVHDPIDAEDPASADLLHAITLELEKQAWMIGSENRSPRRggsgggsgggsggsgKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 160 rTT-NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN dps(kr) 1NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsggTTIHDVQTTGLTQDAVTGFDASSRLNAGLQEVLVDLTALHLQGKQAHWNIVGENWRDLHLQLDTLVEAARGFSDDVAERMRAVGGVPDARPQTVAASRIGDVGPDEIDTRACVEAIVALVRHTVDTIRRVHDPIDAEDPASADLLHAITLELEKQAWMIGSENRS PRRR 161rTT- NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNdps(kr) 2 NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsggTTIHDVQTTGLTQDAVTGFDASSRLNAGLQEVLVDLTALHLQGKQAHWNIVGENWRDLHLQLDTLVEAARGFSDDVAERMRAVGGVPDARPQTVAASRIGDVGPDEIDTRACVEAIVALVRHTVDTIRRVHDPIDAEDPASADLLHAITLELEKQAWMIGS ENRSPRRR162 dps(np)-SETQTLLRNFGNVYDNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLH rTT 1EFFNEAYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQMVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLSHFLAKDSLTLGFVQAAQSggsggKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKD WTND 163dps(np)- SETQTLLRNFGNVYDNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLHrTT 2 EFFNEAYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQMVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLSHFLAKDSLTLGFVQAAQSggsgggsggKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 164 dps(np)-SETQTLLRNFGNVYDNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLH rTT 3EFFNEAYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQMVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLSHFLAKDSLTLGFVQAAQSggsgggsgggsggsgKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 165 rTT-NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN dps(np) 1NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsggMSETQTLLRNFGNVYDNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLHEFFNEAYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQMVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLSHFLAKDSLTLGFVQAAQS 166 rTT-NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN dps(np) 2NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsggMSETQTLLRNFGNVYDNPVLLDRSVTAPVTEGFNVVLASFQALYLQYQKHHFVVEGSEFYSLHEFFNEAYNQVQDHIHEIGERLDGLGGVPVATFSKLAELTCFEQESEGVYSSRQMVENDLAAEQAIIGVIRRQAAQAESLGDRGTRYLYEKILLKTEERAYHLSHFLAKDSLTLGFVQAAQS Bacteriophage Q Beta Capsid, Chain A 167rTT-qbeta NLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN1 NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsggsgsggAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANG

CDPSVTRQAYADVTFSFTQ YSTDEERAFVRTELAALLASPLLIDAIDQLNPAY 168 rTT-qbetaNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 2NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggdgssgdggAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANG

CDPSVTRQAYADVTFSFTQ YSTDEERAFVRTELAALLASPLLIDAIDQLNPAY 169 rTT-qbetaNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 3NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgggsgggsggsgAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANG

CDPSVTRQAYADVT FSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY 170 rTT-qbetaNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 4NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggdsggdgggdggdgAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANG

CDPSVTRQAYADVT FSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY 171 rTT-qbetaNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVN 5NEASEVIVHKAMDIEYNDMFNQFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsggppppgsggsgAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANG

CDPSVTRQAYADVT FSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAYCaMKIIa (12-mer) C-term fragment (5U6Y) 172 CRM-5U6YGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA 1AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSggksggnkksdgvkessestntaiededtkvrkqeiikvteqlieaisngdfesytkmcdpgmtafepealgnlvegldfhrfyfenlwsrnskpvhttilnphihlmgdesaciayiritqyldaggiprtaqseetrvwhrrdgkwqivhfhrsga 173 CRM-5U6YGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA 2AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgvkessestntaiededtkvrkqeiikvteqlieaisngdfesytkmcdpgmtafepealgnlvegldfhrfyfenlwsrnskpvhttilnphihlmgdesaciayiritqyldaggiprtaqseetrvwhrrdgkwqivhfhrsga 174CRM-5U6Y GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA3 AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgstntaiededtkvrkqeiikvteqlieaisngdfesytkmcdpgmtafepealgnlvegldfhrfyfenlwsrnskpvhttilnphihlmgdesaciayiritqyldaggiprtaqseetrvwhrrdgkwqivhfhsga 175 HID-5U6YSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHF 1LDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIKggksggnkksdgvkessestntaiededtkvrkqeiikvteqlieaisngdfesytkmcdpgmtafepealgnlvegldfhrfyfenlwsrnskpvhttilnphihlmgdesaciayiritqyldaggiprtaqseetrvwhrrdgkwqivhfhr sga 176HID-5U6Y SNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHF2 LDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIKgvkessestntaiededtkvrkqeiikvteqlieaisngdfesytkmcdpgmtafepealgnlvegldfhrfyfenlwsrnskpvhttilnphihlmgdesaciayiritqyldaggiprtaqseetrvwhrrdgkwqivhnrsga 177 HID-5U6YSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHF 3LDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIKgstntaiededtkvrkqeiikvteqlieaisngdfesytkmcdpgmtafepealgnlvegldfhrfyfenlwsrnskpvhttilnphihlmgdesaciayiritqyldaggiprtagseetrvwhrrdgkwqivhfhrsgaPhosphopantetheine Adenylyltransferase (6ccq) 178 CRM-6CCQ-GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDA rTTAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSggggsggggsMQKRAIYPGTFDPITNGHIDIVTRATQMFDHVILAIAASPSKKPMFTLEERVALAQQATAHLGNVEVVGFSDLMANFARNQHATVLIRGLRAVADFEYEMQLAHMNRHLMPELESVFLMPSKEWSFISSSLVKEVARHQGDVTHFLPENVHQALMAKLAVDggggsggggsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 179 HID-6CCQ-SNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHF rTTLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIKggggsggggsMQKRAIYPGTFDPITNGHIDIVTRATQMFDHVILAIAASPSKKPMFTLEERVALAQQATAHLGNVEVVGFSDLMANFARNQHATVLIRGLRAVADFEYEMQLAHMNRHLMPELESVFLMPSKEWSFISSSLVKEVARHQGDVTHFLPENVHQALMAKLAVDggggsggggsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDT4 fibritin Foldon domain (Fd) 180 Fd-GYIPEAPRDGQAYVRKDGEWVLLSTFLGSGGGGGQMKNLDCWVDNEEDIDVILKKSTILNLD rTT_deglyINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 181 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL Fd-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI TT_deglyGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGGGGGQMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 182 Fd-GYIPEAPRDGQAYVRKDGEWVLLSTFLGSGGGGGQMKNLDCWVDkEEDIDVILKKSTILNLDrTT_degly-INkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTV K20SFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 183 rTT_degly-MKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHL Fd-VNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSI TT_degly-GSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYIN K20GVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGGGGGQMKNLDCWVDkEEDIDVILKKSTILNLDINkDIISDISkFNSAVITYPDAQLVPGINGKAIHLVNNEkSEVIVHKAMkIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHkkSIGSGWSVSLKGNNLIWTLKDSkGEVRQITFRDLPkKFNAYLANKWVFITITNDRkSSANLYINGVLMGSAEITkLGAIREDNQITLKLDRCkNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTkAPSYTNGKLNIYYRRLYNGLKFIIKRYkPNNkIDSFVKSGDFIKLYVSYkNNEHIVGYPKDGNAFNkLDRILRVGYkAPkIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGkDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND Hexamer 184 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLr8_linker-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggaeaaakeaaakeaaakeaaakaleaeaaakeaaakeaaakeaaakaEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 185 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL12pa_linker-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgspapapapapapapapapapapapaEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 186 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLr3_linker-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggaeaaakeaaakeaaakaEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 187 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL5gA_linker-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgsgsgsgsasgasgEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 188 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL3f_linker-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 189 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL2rf_linKer-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsaeaaakeaaakaEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 190 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL1f_linker-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsasgEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY 191 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL1rf_liner-VNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI Fc-GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYIN HexamerGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDpapapasgEPKSCDKTHTCPKCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVCLQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKggsggPTLYNVSLVMSDTAGTCY DIHYDROLIPOYL TRANSACETYLASE (e2p) 192CRM-e2p 1 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKggggsggggsGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 193 HID-e2pSNMANTQMKSDKIIIAHRGASGYLPEHTLESKALAFAQQADYLEQDLAMTKDGRLVVIHDHFLDGLTDVAKKFPHRHRKDGRYYVIDFTLKEIQSLEMTENFETKDGKQAQVYPNRFPLWKSHFRIHTFEDEIEFIQGLEKSTGKKVGIYPEIKAPWFHHQNGKDIAAETLKVLKKYGYDKKTDMVYLQTFDFNELKRIKTELLPQMGMDLKLVQLIAYTDWKETQEKDPKGYWVNYNYDWMFKPGAMAEVVKYADGVGPGWYMLVNKEESKPDNIVYTPLVKELAQYNVEVHPYTVRKDALPEFFTDVNQMYDALLNKSGATGVFTDFPDTGVEFLKGIKggggsggggsGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSA LREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 194 CRM-e2p 2GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSggggsggggsGAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 195 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 10f-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsggggsaeaaakeaaakaggggsggggsggggsggggsAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELL LM 196rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 8f-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsggggsggggsggggsggggsggggsAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 197 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 4f-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsggggsAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREY PVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 198 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 3f-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsggggsAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 199 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 2f-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsggggsAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDE TEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 200 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 1f-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggggsasgAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETE EIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 201 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL r8-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggaeaaakeaaakeaaakeaaakaleaeaaakeaaakeaaakeaaakasgAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 202rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL r12-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsapapapapapapapapapapapapasgAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 203 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL r3-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgaeaaakeaaakeaaakasgAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREY PVLNT

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM 204 rTT_degly-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHL 2rf-E2pVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsaeaaakeaaakasgAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLN T

IDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM Glutamate Synthase, Chain A (1f52) 2056H-3C- SnmantqmksdkiiiahrgasgylpehtleskalafaqqadyleqdlamtkdgrlvvihdhfHiD-1f52 ldgltdvakkfphrhrkdgryyvidftlkeigslemtenfetkdgkqaqvypnrfplwkshfrihtfedeiefigglekstgkkvgiypeikapwfhhqngkdiaaetlkylkkygydkktdmvylqtfdfnelkriktellpqmgmdlklvqliaytdwketqekdpkgywynynydwmfkpgamaevykyadgvgpgwymlynkeeskpdnivytplykelagynvevhpytyrkdalpefftdvnqmydallnksgatgvftdfpdtgveflkgikSAEHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDRVRMTPHPVEFELY YSV 2066H-3C- MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLrTT-1f52 VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDSAEHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDRVRMTPHPVEFELYYSV 207 6H-3C-MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHL rTT- ln4-VNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSI 1f52GSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgggsSAEHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDRVRMTPHPVEFELYYSV 208 6H-3C-GADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDACRM_degly-AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFI 1f52KRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSSAEHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDRVRMTPHPVEFELYYSV 209 6H-3C-GADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDACRM_degly-AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFI ln4-1f52KRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgggsSAEHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDRVRMTPHPVEFELYYSV 210 6H-3C-GADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDACRM_degly-AGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFI ln8-1f52KRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgggsgggsSAEHVLTMLNEHEVKFVDLRFTDTKGKEQHVTIPAHQVNAEFFEEGKMFDGSSIGGWKGINESDMVLMPDASTAVIDPFFADSTLIIRCDILEPGTLQGYDRDPRSIAKRAEDYLRATGIADTVLFGPEPEFFLFDDIRFGASISGSHVAIDDIEGAWNSSTKYEGGNKGHRPGVKGGYFPVPPVDSAQDIRSEMCLVMEQMGLVVEAHHHEVATAGQNEVATRFNTMTKKADEIQIYKYVVHNVAHRFGKTATFMPKPMFGDNGSGMHCHMSLAKNGTNLFSGDKYAGLSEQALYYIGGVIKHAKAINALANPTTNSYKRLVPGYEAPVMLAYSARNRSASIRIPVVASPKARRIEVRFPDPAANPYLCFAALLMAGLDGIKNKIHPGEPMDKNLYDLPPEEAKEIPQVAGSLEEALNALDLDREFLKAGGVFTDEAIDAYIALRREEDDRVRMTPHPVEFELYYSV HIV capsid oligerization domain (HIV) 211 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTTQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 212HIV-CA- PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH3P0A-rTT- QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI858 PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTD EGWTND213 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTT-QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI 836PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVskfigitelkkleskinkvfsTpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 214 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTT-QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI 217-839PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTAigitelkkleskinkvfsTpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 215 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTT-QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI 217-840PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTAgitelkkleskinkvfsTpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 216 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTT-QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI 217-841PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTAitelkkleskinkvfsTpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 217 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTT-QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI 217-842PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTAtelkkleskinkvfsTpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFV PTDEGWTND218 HIV-CA-PIVQNLQGQMVHQAISCLCLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGH 3P0A-rTT-QAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPI 217-843PVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTAtelkkleskinkvfsTpipfsysKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFV PTDEGWTND219 HIV-CA-PIVQNLQGQMVHQCISPRTLNAWVKVVEEKAFSPEVIPMFSALSCGATPQDLNTMLNTVGGH 3H4E-rTTQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNAATETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDEncapsulin 254 EN-TThc-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN deglyEVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgsgsgsMKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSAVITYPDAQLVPGINGKAIHLVNNEASEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNQITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKQITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKQASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND 255 EN-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN CRM197-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCE deglyKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgsgsgsGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS 256 EN-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN 2xCRM197-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCE deglyKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgsgDYKDDDDKgsgGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSgggsgggsGADDVVDSSKSFVMENFASYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKAVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKAKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS 257 EN-HIDMEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgsgDYKDDDDKgsgsnmantqmksdkiiiahrgasgylpehtleskalafaqqadyleqdlamtkdgrlvvihdhfldgltdvakkfphrhrkdgryyvidftlkeiqslemtenfetkdgkqaqvypnrfplwkshfrihtfedeiefiqglekstgkkvgiypeikapwfhhqngkdiaaetlkvlkkygydkktdmvylqtfdfnelkriktellpqmgmdlklvqliaytdwketqekdpkgywynynydwmfkpgamaevvkyadgvgpgwymlvnkeeskpdnivytplvkelaqynvevhpytvrkdalpefftdvnqmydallnksgatgvftdfpdtgveflkgik 369 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C-KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTTHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgggsgggsgggsKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnftvsfwlrvpkvsashleqygtneysiissmkkhslsigsgwsyslkgnnliwtlkdsagevrqitfrdlpdkfnaylankwvfititndrlssanlyingvlmgsaeitglgairednnitlkldrcnnnnqyvsidkfrifckalnpkeieklytsylsitflrdfwgnplrydteyylipvassskdvqlknitdymyltnapsytngklniyyrrlynglkfiikrytpnneidsfvksgdfiklyvsynnnehivgypkdgnafnnldrilrvgynapgiplykkmeavklrdlktysvqlklyddknaslglvgthngqigndpnrdiliasnwyfnhlkdkilgcdwyfvptdegwtnd 370 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C -KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTTHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgggsgggsgggslrdfwgnplrydteyylipvassskdvqlknitdymyltnapsytngklniyyrrlynglkfiikrytpnneidsfvksgdfiklyvsynnnehivgypkdgnafnnldrilrvgynapgiplykkmeavklrdlktysvqlklyddknaslglvgthngqigndpnrdiliasnwyfnhlkdkilgcdwyfvptdegwtnd 371 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C -KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTTHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITE N249TFTFQVVNPEALILLKFgggsgggsgggsknldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnftvsfwlrvpkvsashleqygtneysiissmkkhslsigsgwsyslkgnnliwtlkdsagevrqitfrdlpdkfnaylankwvfititndrlssanlyingvlmgsaeitglgairednnitlkldrcnnnnqyvsidkfrifckalnpkeieklytsylsit 372 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C -KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTTHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITE N193TFTFQVVNPEALILLKFgggsgggsgggsknldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnftvsfwlrvpkvsashleqygtneysiissmkkhslsigsgwsvslkgnnliwtlkdsagevrqitfrdlpdkfnaylankwvfititndrlssanlyingvlmgsae 373 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C -KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTT N87HYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgggsgggsgggsKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 374 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLErGcPNVDLSSLEETVRKVAEFEDEVIFRGCEK146/A185C -KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTTHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITE N87TFTFQVVNPEALILLKFgggsgggsgggsKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 375 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLgEVEVLSDEN glyser-EVVKWGLRKSLPLIELRATFTLDLWELDNLErGkPNVDLSSLEETVRKVAEFEDEVIFRGCE G53C/K96CKSGVKGLLSFEERKIECGSTPcDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEcG rTT N87HYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgggsgggsgggsKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 376 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN caIgG2a-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C -KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTT N88HYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFggEPKIPQPQPKPQPQPQPQPKPQPKPEPEggKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 377 EN -CD8-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDENG53C/K96C-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCE rTT N88KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFggKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDggKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 378 EN -MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDEN hinge-EVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEG53C/K96C-KSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAG rTT N88HYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFggEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLggKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf HBV 379 rTT-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV hinge-HBVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGP25C/R127CSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMDIDPYKEFGATVELLSFLPSDFFcSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 380 rTT-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV hinge-HBVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIG E14C/A36CSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMDIDPYKEFGATVcLLSFLPSDFFPSVRDLLDTAScLYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 381 rTT-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV hinge-HBVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGD29C/R127CSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDggsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsggMDIDPYKEFGATVELLSFLPSDFFPSVRcLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 382 rTT-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV caIgG2a-NNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIG HBVSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGP25C/R127CVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEPEgsgMDIDPYKEFGATVELLSFLPSDFFcSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 383 rTT-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV caIgG2a-NNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIG HBVSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYING E14C/A36CVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEPEgsgMDIDPYKEFGATVcLLSFLPSDFFPSVRDLLDTAScLYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 384 rTT-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV caIgG2a-NNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIG HBVSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGD29C/R127CVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgGSEPKIPQPQPKPQPQPQPQPKPQPKPEPEgsgMDIDPYKEFGATVELLSFLPSDFFPSVRcLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC 385 rTT-CD8-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV HBVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGP25C/R127CSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsggsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMDIDPYKEFGATVELLSFLPSDFFcSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRR SQSRESQC386 rTT-CD8-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV HBVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIG E14C/A36CSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsggsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMDIDPYKEFGATVcLLSFLPSDFFPSVRDLLDTAScLYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRR SQSRESQC387 rTT-CD8-KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLV HBVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGD29C/R127CSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsggsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgMDIDPYKEFGATVELLSFLPSDFFPSVRcLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIcTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRR SQSRESQCAP205 388 AP205-MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKP glyser-EGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI rTTVSSDTTgggsgggsgggsKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnftvsfwlrvpkvsashleqygtneysiissmkkhslsigsgwsyslkgnnliwtlkdsagevrqitfrdlpdkfnaylankwvfititndrlssanlyingvlmgsaeitglgairednnitlkldrcnnnnqyvsidkfrifckalnpkeieklytsylsitflrdfwgnplrydteyylipvassskdvqlknitdymyltnapsytngklniyyrrlynglkfiikrytpnneidsfyksgdfiklyvsynnnehivgypkdgnafnnldrilrvgynapgiplykkmeavklrdlktysvqlklyddknaslglvgthngqigndpnrdiliasnwyfnhlkdkilgcdwyfvptdegwtnd 389 AP205-MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKP glyser-EGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI rTT N193VSSDTTgggsgggsgggsknldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnftvsfwlrvpkvsashleqygtneysiissmkkhslsigsgwsyslkgnnliwtlkdsagevrqitfrdlpdkfnaylankwvfititndrlssanlyingvlmgsae 390 AP205-MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKP glyser-EGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI rTT N87VSSDTTgggsgggsgggsKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 391 AP205-MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKP caIgG2a-EGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI rTT N87VSSDTTgsgEPKIPQPQPKPQPQPQPQPKPQPKPEPEgsgKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfn nf 392AP205- MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPCD8-rTT EGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAIN87 VSSDTTgsgKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDgsgKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdiey ndmfnnf393 AP205-MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKP hinge-rTTEGCADACVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI N87VSSDTTgsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsgKnldcwvdneedidvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 394 AP205MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKP T81C-EGCADACVIMPNENQSIRcVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI hinge-rTTVSSDTTgsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsgKnldcwvdneedi N87dvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 395 AP205MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVVYcKRPAPKPS53C/H100C-EGCADACVIMPNENQSIRTVISGSAENLATLKAEWETcKRNVDTLFASGNAGLGFLDPTAAI hinge-VSSDTTgsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsgKnldcwvdneedi rTT N87dvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 396 AP205MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPV82C/R80C-EGCADACVIMPNENQSIctcISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAAI hinge-VSSDTTgsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsgKnldcwvdneedi rTT N87dvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf 397 AP205-MANKPMQPITSTANKIVWSDPTRLSTTFSASLLRQRVKVGIAELNNVSGQYVSVYKRPAPKPC65/C69GC-EGCADAgCVIMPNENQSIRTVISGSAENLATLKAEWETHKRNVDTLFASGNAGLGFLDPTAA hinge-IVSSDTTgsgEPKSDKTHTPPPAPELLgsgEPKSDKTHTPPPAPELLgsgKnldcwvdneed rTT N87idvilkkstilnldinndiisdisgfnssvitypdaqlvpgingkaihlvnnessevivhkamdieyndmfnnf

indicates data missing or illegible when filed

The fusion protein of the self-assembling protein nanoparticle carriercan include various tags and sequences for production and purification.Typically such protein tags are linked to the N- or C-terminus of themonomer and are ultimately removed (for example by selective proteasecleave) from the monomer. For production in cells, the fusion protein ofthe self-assembling protein nanoparticle carrier can further include asignal peptide that is cleaved off during cellular processing. Thefusion proteins can be expressed in appropriate cells (e.g., HEK 293Freestyle cells) and the fusion proteins are secreted from the cells andself-assemble into the protein nanoparticle carrier. The proteinnanoparticle carrier can be purified using known techniques, for exampleby a few different chromatography procedures, e.g. Mono Q (anionexchange) followed by size exclusion (SUPEROSE® 6) chromatography.

B. HIV-1 Env Fusion Peptide

Any combination of HIV-1 Env fusion peptide and self-assembling proteinnanoparticle carrier may be selected from the specific HIV-1 Env fusionpeptide and self-assembling protein nanoparticle carriers providedherein to generate the immunogenic conjugate.

HIV-1 can be classified into four groups: the “major” group M, the“outlier” group 0, group N, and group P. Within group M, there areseveral genetically distinct clades (or subtypes) of HIV-1. The HIV-1Env fusion peptide included in the immunogenic conjugate can be thefusion peptide from any subtype of HIV, such as groups M, N, O, or P orclade A, B, C, D, F, G, H, J or K and the like.

The HIV-1 Env fusion peptide included in the immunogenic conjugate canconsist essentially of or consist of residue 512 to one of residues514-521 (such as residues 512-519) of HIV-1 Env (HXB2) numbering of theEnv protein from any subtype of HIV, such as groups M, N, O, or P orclade A, B, C, D, F, G, H, J or K and the like. In some embodiments, theHIV-1 Env fusion peptide included in the immunogenic conjugate canconsist essentially of or consist of residue 512 to one of residues515-521 of HIV-1 Env (HXB2) numbering of the Env protein from anysubtype of HIV, such as groups M, N, O, or P or clade A, B, C, D, F, G,H, J or K and the like. In some embodiments, The HIV-1 Env fusionpeptide included in the immunogenic conjugate can consist essentially ofor consist of residue 512 to one of residues 516-521 of HIV-1 Env (HXB2)numbering of the Env protein from any subtype of HIV, such as groups M,N, O, or P or clade A, B, C, D, F, G, H, J or K and the like. HIV Envfusion peptides from the different HIV Glades, as well as nucleic acidsequences encoding such proteins and methods for the manipulation andinsertion of such nucleic acid sequences into vectors, are known (see,e.g., HIV Sequence Compendium, Division of AIDS, National Institute ofAllergy and Infectious Diseases (2003); HIV Sequence Database(hiv-web.lanl.gov/content/hiv-db/mainpage.html); Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989); Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994)).

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGIGAVFLG (SEQ ID NO: 1). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO:1.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGLGAVFLG (SEQ ID NO: 2). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 2.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGIGAMIFG (SEQ ID NO: 3). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 3.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to11 residues (such as 5, 6, 7, 8, 9, 10, or 11 residues or 7-9 residuesor 8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGTIGAMFLG (SEQ ID NO: 4). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 4.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGIGAMFLG (SEQ ID NO: 5). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 5.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGIGALFLG (SEQ ID NO: 6). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 6.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AIGLGAMFLG (SEQ ID NO: 7). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 7.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGLGAVFIG (SEQ ID NO: 8). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 8.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGIGAVLLG (SEQ ID NO: 9). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 9.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AVGIGAVFIG (SEQ ID NO: 10). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 10.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to10 residues (such as 5, 6, 7, 8, 9, or 10 residues or 7-9 residues or8-10 residues or 6-8 residues) from the N-terminus of the amino acidsequence set forth as AIGLGALFLG (SEQ ID NO: 11). These residuescorrespond to HIV-1 Env positions 512-521 (HXB2 numbering). In someembodiments, the HIV-1 Env fusion peptides included in the immunogenicconjugate consists essentially of or consists of the amino acid sequenceset forth as residues 1-8 of SEQ ID NO: 11.

In some embodiments, the HIV-1 Env fusion peptides included in theimmunogenic conjugate consists essentially of or consists of from 5 to 9residues (such as 5, 6, 7, 8, or 9 residues or 7-9 residues or 8-9residues or 6-8 residues) from the N-terminus of the amino acid sequenceset forth as AALGAVFLG (SEQ ID NO: 12). These residues correspond toHIV-1 Env positions 512-521 (HXB2 numbering). In some embodiments, theHIV-1 Env fusion peptides included in the immunogenic conjugate consistsessentially of or consists of the amino acid sequence set forth asresidues 1-8 of SEQ ID NO: 12.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising a tetanus toxoid heavy chain Cfragment and a lumazine synthase nanoparticle subunit, wherein the HIV-1Env fusion peptides are conjugated to the self-assembling proteinnanoparticle carrier by linkers between lysine residues on theself-assembling protein nanoparticle carrier and a heterologous cysteineresidue fused to a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising H. influenzae protein D (HiD) anda lumazine synthase nanoparticle subunit, wherein the HIV-1 Env fusionpeptides are conjugated to the self-assembling protein nanoparticlecarrier by linkers between lysine residues on the self-assemblingprotein nanoparticle carrier and a heterologous cysteine residue fusedto a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising diphtheria toxoid or a variantthereof (such as CRM197) and a lumazine synthase nanoparticle subunit,wherein the HIV-1 Env fusion peptides are conjugated to theself-assembling protein nanoparticle carrier by linkers between lysineresidues on the self-assembling protein nanoparticle carrier and aheterologous cysteine residue fused to a C-terminal residue of the HIV-1Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising a tetanus toxoid heavy chain Cfragment and a ferritin nanoparticle subunit, wherein the HIV-1 Envfusion peptides are conjugated to the self-assembling proteinnanoparticle carrier by linkers between lysine residues on theself-assembling protein nanoparticle carrier and a heterologous cysteineresidue fused to a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising H. influenzae protein D (HiD) anda ferritin nanoparticle subunit, wherein the HIV-1 Env fusion peptidesare conjugated to the self-assembling protein nanoparticle carrier bylinkers between lysine residues on the self-assembling proteinnanoparticle carrier and a heterologous cysteine residue fused to aC-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising diphtheria toxoid or a variantthereof (such as CRM197) and a ferritin nanoparticle subunit, whereinthe HIV-1 Env fusion peptides are conjugated to the self-assemblingprotein nanoparticle carrier by linkers between lysine residues on theself-assembling protein nanoparticle carrier and a heterologous cysteineresidue fused to a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising a tetanus toxoid heavy chain Cfragment and a lumazine synthase nanoparticle subunit and furthercomprising a heterologous T-cell helper epitope (such as AENLWVTVYYGVPVW(SEQ ID NO: 70) or TEKLWVTVYYGVPVW (SEQ ID NO: 71), wherein the HIV-1Env fusion peptides are conjugated to the self-assembling proteinnanoparticle carrier by linkers between lysine residues on theself-assembling protein nanoparticle carrier and a heterologous cysteineresidue fused to a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising H. influenzae protein D (HiD) anda lumazine synthase nanoparticle subunit and further comprising aheterologous T-cell helper epitope (such as AENLWVTVYYGVPVW (SEQ ID NO:70) or TEKLWVTVYYGVPVW (SEQ ID NO: 71), wherein the HIV-1 Env fusionpeptides are conjugated to the self-assembling protein nanoparticlecarrier by linkers between lysine residues on the self-assemblingprotein nanoparticle carrier and a heterologous cysteine residue fusedto a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising diphtheria toxoid or a variantthereof (such as CRM197) and a lumazine synthase nanoparticle subunitand further comprising a heterologous T-cell helper epitope (such asAENLWVTVYYGVPVW (SEQ ID NO: 70) or TEKLWVTVYYGVPVW (SEQ ID NO: 71),wherein the HIV-1 Env fusion peptides are conjugated to theself-assembling protein nanoparticle carrier by linkers between lysineresidues on the self-assembling protein nanoparticle carrier and aheterologous cysteine residue fused to a C-terminal residue of the HIV-1Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising a tetanus toxoid heavy chain Cfragment and a ferritin nanoparticle subunit and further comprising aheterologous T-cell helper epitope (such as AENLWVTVYYGVPVW (SEQ ID NO:70) or TEKLWVTVYYGVPVW (SEQ ID NO: 71), wherein the HIV-1 Env fusionpeptides are conjugated to the self-assembling protein nanoparticlecarrier by linkers between lysine residues on the self-assemblingprotein nanoparticle carrier and a heterologous cysteine residue fusedto a C-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising H. influenzae protein D (HiD) anda ferritin nanoparticle subunit and further comprising a heterologousT-cell helper epitope (such as AENLWVTVYYGVPVW (SEQ ID NO: 70) orTEKLWVTVYYGVPVW (SEQ ID NO: 71), wherein the HIV-1 Env fusion peptidesare conjugated to the self-assembling protein nanoparticle carrier bylinkers between lysine residues on the self-assembling proteinnanoparticle carrier and a heterologous cysteine residue fused to aC-terminal residue of the HIV-1 Env fusion peptides.

In some embodiments, the immunogenic conjugate comprises any of theabove HIV-1 Env fusion peptides (such as AVGIGAVF, residues 1-8 of SEQID NO: 1) conjugated to a self-assembling protein nanoparticle carrierformed from fusion proteins comprising diphtheria toxoid or a variantthereof (such as CRM197) and a ferritin nanoparticle subunit and furthercomprising a heterologous T-cell helper epitope (such as AENLWVTVYYGVPVW(SEQ ID NO: 70) or TEKLWVTVYYGVPVW (SEQ ID NO: 71), wherein the HIV-1Env fusion peptides are conjugated to the self-assembling proteinnanoparticle carrier by linkers between lysine residues on theself-assembling protein nanoparticle carrier and a heterologous cysteineresidue fused to a C-terminal residue of the HIV-1 Env fusion peptides.

Typically, the HIV-1 Env fusion peptides are conjugated to theself-assembling protein nanoparticle carrier by a linker. Suitablelinkers include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers or peptide linkers. For animmunogenic conjugate from two or more constituents, each of theconstituents will contain the necessary reactive groups. Representativecombinations of such groups are amino with carboxyl to form amidelinkages or carboxy with hydroxyl to form ester linkages or amino withalkyl halides to form alkylamino linkages or thiols with thiols to formdisulfides or thiols with maleimides or alkylhalides to form thioethers.Hydroxyl, carboxyl, amino and other functionalities, where not presentmay be introduced by known methods. Likewise, a wide variety of linkinggroups may be employed. In some cases, the linking group can be designedto be either hydrophilic or hydrophobic in order to enhance the desiredbinding characteristics of the fusion peptide and the carrier. Thecovalent linkages should be stable relative to the solution conditionsunder which the conjugate is subjected.

In some embodiments, the linkers may be joined to the constituent aminoacids through their side chains (such as through a disulfide linkage tocysteine) or to the alpha carbon, amino, and/or carboxyl groups of theterminal amino acids.

The procedure for attaching a molecule to a polypeptide varies accordingto the chemical structure of the molecule. Polypeptides typicallycontain a variety of functional groups; for example, carboxylic acid(COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on apolypeptide. Alternatively, the polypeptide is derivatized to expose orattach additional reactive functional groups. The derivatization mayinvolve attachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill.

In some embodiments, a sulfosuccinimidyl (4-iodoacetyl)aminobenzoate(Sulfo-SIAB) linker is used to link the HIV-1 Env fusion peptides to theself-assembling protein nanoparticle carrier. In some embodiments anm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) linker is used tolink the HIV-1 Env fusion peptides to the self-assembling proteinnanoparticle carrier.

The immunogenic conjugate includes a plurality of HIV-1 Env fusionpeptides conjugated to the self-assembling protein nanoparticle carrier.In several embodiments, the conjugation of multiple HIV-1 Env fusionpeptides to a single self-assembling protein nanoparticle carrier ispossible because the carrier has multiple lysine or cysteine side-chainsthat can serve as sites of attachment. The amount of HIV-1 Env fusionpeptide reacted with the amount of self-assembling protein nanoparticlecarrier may vary depending upon the specific HIV-1 Env fusion peptideand the self-assembling protein nanoparticle carrier. The resultingnumber of HIV-1 Env fusion peptides linked to a single self-assemblingprotein nanoparticle carrier molecule may vary depending upon thespecific HIV-1 Env fusion peptides and the self-assembling proteinnanoparticle carrier.

Following conjugation of the HIV-1 Env fusion peptide to the proteinnanoparticle carrier, the conjugate can be purified by appropriatetechniques. One goal of the purification step is to separate theunconjugated HIV-1 Env fusion peptide or protein nanoparticle carrierfrom the conjugate. One method for purification, involvingultrafiltration in the presence of ammonium sulfate, is described inU.S. Pat. No. 6,146,902. Alternatively, the conjugates can be purifiedaway from unconjugated HIV-1 Env fusion peptide or protein nanoparticlecarrier by any number of standard techniques including, for example,size exclusion chromatography, density gradient centrifugation,hydrophobic interaction chromatography, or ammonium sulfatefractionation. See, for example, Anderson et al., J. Immunol.137:1181-86, 1986 and Jennings & Lugowski, J. Immunol. 127:1011-18,1981. The compositions and purity of the conjugates can be determined byGLC-MS and MALDI-TOF spectrometry, for example.

In several embodiments, the disclosed immunogenic conjugates can beformulated into immunogenic composition (such as vaccines), for exampleby the addition of a pharmaceutically acceptable carrier and/oradjuvant.

It is understood that some variations can be made in the amino acidsequence of a protein without affecting the activity of the protein.Such variations include insertion of amino acid residues, deletions ofamino acid residues, and substitutions of amino acid residues. Thesevariations in sequence can be naturally occurring variations or they canbe engineered through the use of genetic engineering techniques.Examples of such techniques are found in see, e.g., Sambrook et al.(Molecular Cloning: A Laboratory Manual, 4^(th) ed, Cold Spring Harbor,N.Y., 2012) and Ausubel et al. (In Current Protocols in MolecularBiology, John Wiley & Sons, New York, through supplement 104, 2013, bothof which are incorporated herein by reference in their entirety. Thus,the sequence of the fusion proteins of the disclosed self-assemblingprotein nanoparticle carrier can include modifications, such as aminoacid substitutions, deletions or insertions, glycosylation and/orcovalent linkage to unrelated proteins (e.g., a protein tag), as long asthe fusion proteins self-assemble to form the self-assembling proteinnanoparticle carrier.

III. NANOPARTICLES LINKED TO CARRIER BY ISOPEPTIDE BOND

Also provided herein are embodiments of a self-assemblingnanoparticle-carrier protein where the nanoparticle is linked to carrierproteins by an isopeptide bond between a first tag on the subunits ofthe nanoparticle and a second tag on the carrier protein. In oneexample, the first and second tags are based on the Streptococcuspyogenes fibronectin binding protein Fbab-B, such as in theSpyTag/SpyCatcher fusion system. The sequence of Streptococcus pyogenesfibronectin binding protein Fbab-B is provided as follows:

(SEQ ID NO: 398) GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDIDGKELAGATMELRDSSGKTISTWISDGQVKDFYLMPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIVMVDAYKPTK

The final 13 residues of Streptococcus pyogenes fibronectin bindingprotein Fbab-B can be used as a first tag (the spytag) and the remainingresidues of Streptococcus pyogenes fibronectin binding protein Fbab-Bare the second tag (spycatcher). When mixed under appropriate conditionsthe two Streptococcus pyogenes fibronectin binding protein Fbab-Bsegments bind and form a covalent isopeptide bond.

Any of the nanoparticle subunits disclosed herein can be linked to anyof the carrier proteins disclosed herein using the spytag/spycatcher (orother suitable isopeptide bond tag) to generate a nanoparticle-carrierprotein to which one or more vaccine antigens (such as an HIV-1 Envfusion peptide as disclosed herein) can be conjugated. In severalembodiments, the spytag/spycatcher (or other suitable isopeptide bondtag) is substituted for the peptide linker separating the nanoparticlesubunit and carrier protein.

In some embodiments, a lumazine synthase subunit is fused to a spytagand combined with any of the carrier proteins described herein that hasbeen fused to a corresponding spycatcher tag. Non-limiting examples oflumazine synthase subunits fused to a spytag for use in the disclosedembodiments, include:

LS-SpyTag (SEQ ID NO: 399)AHIVMVDAYKPTKgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR LS-SpyTag LODS3 (single Cysteine?)(SEQ ID NO: 400) AHIVMVDAYKPTKgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGcIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR LS-SpyTag LODS5 (intra-protomer) (SEQ ID NO: 401)AHIVMVDAYKPTKgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPIcFGVITADTLEQAIERAGTKHGNKGWEAALcAIEMANLFKSLR LS-SpyTag DS2-49 (SEQ ID NO: 402)AHIVMVDAYKPTKgsgsaMcIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDIcLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR LS-SpyTag DS54-142 (SEQ ID NO: 403)AHIVMVDAYKPTKgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVcGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALcAIEMANLFKSLR LS-SpyTag D595-101 (SEQ ID NO: 404)AHIVMVDAYKPTKgsgsaMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEnIsAVIAIGVLIRGATPHFDYIAScVSKGLcDLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR

In some embodiments, a ferritin subunit is fused to a spytag andcombined with any of the carrier proteins described herein that has beenfused to a corresponding spycatcher tag. Non-limiting examples offerritin subunits fused to a spytag for use in the disclosedembodiments, include:

Ferr 96N SpyTag N-2-THS (SEQ ID NO: 405)AHIVMVDAYKPTKgggsgDPMLSKDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQnISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Ferr 148S SpyTag N5-THS(SEQ ID NO: 406) AHIVMVDAYKPTKgggsgDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEsHGLYLADQYVKGIAKSRKS

In some embodiments, an encapsulin subunit is fused to a spytag andcombined with any of the carrier proteins described herein that has beenfused to a corresponding spycatcher tag. A non-limiting examples of anencapsulin subunit fused to a spytag for use in the disclosedembodiments, includes:

EN G53C-R94C - spytag (SEQ ID NO: 410)MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLCEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLECGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKFgggsgAHIVMVDAYKPTK

The spycatcher tag can be genetically fused to any of carrier proteinsprovided herein for subsequent isopeptide bond linkage to a nanoparticlesubunit fused to a corresponding spytag. In some embodiments, a peptidelinker is included between the carrier protein and spycatcher tag orbetween the nanoparticle subunit and spytag. In one example, the rTTcarrier protein fused to the spycatcher tag comprises an amino acidsequence set forth as:

(SEQ ID NO: 407) MKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTNDgsgDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

In one example, the spytag (e.g., AHIVMVDAYKPTK, SEQ ID NO: 408) isgenetically fused to the self-assembling protein nanoparticle subunit,and the nanoparticle with spytag is produced under standard conditions.The spycatcher tag (e.g.,DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI, SEQ ID NO: 409) is genetically fused to thecarrier protein (e.g., rTT), and the carrier protein with spycatcher isproduced under standard conditions. The nanoparticle/spytag andcarrier/spycatcher are subsequently mixed under conditions sufficientfor the spycatcher/spytag to form an isopeptide bond and covalently linkthe nanoparticle and carrier proteins. The resulting nanoparticlecarrier can be used immediately or stored for subsequent conjugation toone or more vaccine antigens of interest.

IV. SELF-ASSEMBLING PROTEIN NANOPARTICLES

Additionally provided herein are novel self-assembling proteinnanoparticles and subunits thereof. In some embodiments, theself-assembling protein nanoparticle subunit comprises or consists ofany one of the self-assembling protein nanoparticle subunit discussedabove in Section II.A.1 for fusion with a heterologous carrier andgeneration of an immunogenic conjugate.

In some embodiments, the self-assembling protein nanoparticle subunit isa lumazine synthase nanoparticle subunit comprising cysteinesubstitutions to introduce one or more non-native disulfide bonds toincrease stability of the nanoparticle, wherein the cysteinesubstitutions comprise 121C and 131C substitutions, 121CG and 131Csubstitutions, 121GC and 131C substitutions, 7C and 40C substitutions,3C and 50C substitutions, 82C and 131CG substitutions, 5C and 52Csubstitutions, or 95C and A101C substitutions, or a combination thereof,wherein residue numbering corresponds to a reference lumazine synthasesubunit set forth as SEQ ID NO: 25. In some embodiments, theself-assembling protein nanoparticle subunit is a lumazine synthasenanoparticle subunit comprising or consisting of the amino acid sequenceset forth as any one of SEQ ID NOs: 306-312, or an amino acid sequenceat least 90% (such as at least 95%, at least 98%, or at least 99%)identical thereto.

In some embodiments, the self-assembling protein nanoparticle subunit isan encapsulin nanoparticle subunit comprising cysteine substitutions tointroduce one or more non-native disulfide bonds to increase stabilityof the nanoparticle, wherein the cysteine substitutions comprise 53C and94C substitutions, 53C and 96C substitutions, or 146C and 185Csubstitutions, or a combination thereof, wherein residue numberingcorresponds to a reference lumazine synthase subunit set forth as SEQ IDNO: 43. In some embodiments, the self-assembling protein nanoparticlesubunit is an encapsulin nanoparticle subunit comprising or consistingof the amino acid sequence set forth as any one of SEQ ID NOs: 313-315,or an amino acid sequence at least 90% (such as at least 95%, at least98%, or at least 99%) identical thereto.

In some embodiments, the self-assembling protein nanoparticle subunit isan Acinetobacter phage AP205 nanoparticle subunit comprising cysteinesubstitutions to introduce one or more non-native disulfide bonds toincrease stability of the nanoparticle, wherein the cysteinesubstitutions comprise a T81C substitution, 53C and 100C substitution,or 82C and 80C substitutions, or a combination thereof, wherein residuenumbering corresponds to a reference lumazine synthase subunit set forthas SEQ ID NO: 316. In some embodiments, the self-assembling proteinnanoparticle subunit is a Acinetobacter phage AP205 protein subunitcomprising or consisting of the amino acid sequence set forth as any oneof SEQ ID NOs: 317-320, or an amino acid sequence at least 90% (such asat least 95%, at least 98%, or at least 99%) identical thereto; or

In some embodiments, the self-assembling protein nanoparticle subunit isa Hepatitis B capsid protein nanoparticle subunit comprising cysteinesubstitutions to introduce one or more non-native disulfide bonds toincrease stability of the nanoparticle, wherein the cysteinesubstitutions comprise 25C and 127C substitutions, 14C and 36Csubstations, 29C and 127C substitutions, 18C and 36C substitutions, or29C and 127C substitutions, or a combination thereof, wherein residuenumbering corresponds to a reference lumazine synthase subunit set forthas SEQ ID NO: 321. In some embodiments, the self-assembling proteinnanoparticle subunit is a Hepatitis B capsid protein subunit comprisingor consisting of the amino acid sequence set forth as any one of SEQ IDNOs: 322-326, or an amino acid sequence at least 90% (such as at least95%, at least 98%, or at least 99%) identical thereto.

In some embodiments, the self-assembling protein nanoparticle subunit isa ferritin nanoparticle subunit comprising or consisting of the aminoacid sequence set forth as any one of SEQ ID NOs: 258-305, or an aminoacid sequence at least 90% (such as at least 95%, at least 98%, or atleast 99%) identical thereto.

In some embodiments, the recombinant self-assembling nanoparticlesubunit is fused to a heterologous carrier protein, such as any of theheterologous carrier proteins discussed above in Section II.A.2 forfusion with a self-assembling protein nanoparticle subunit andgeneration of an immunogenic conjugate. In some embodiments, therecombinant self-assembling nanoparticle subunit is fused to a tetanustoxin heavy chain C fragment, a diphtheria toxin variant CRM197, and anH. influenzae protein D, a Keyhole Limpet Hemocyanin (KLH) functionalunit, a Meningococcal outer membrane protein complex protein, anOuter-membrane lipoprotein carrier protein, or a Cholera toxin Bsubunit. Fusion of the heterologous cattier protein to the recombinantself-assembling nanoparticle subunit can be direct (e.g., vis peptidebond between the nanoparticle subunit and the carrier) or indirect via apeptide linker. Any suitable peptide linker may be used, such as thelinkers discussed above discussed above in Section II.A.3 for fusionwith a self-assembling protein nanoparticle subunit and generation of animmunogenic conjugate.

Also provided are self-assembled protein nanoparticles formed from thenanoparticle subunits. If the nanoparticle subunit is fused to aheterologous carrier protein, then the self-assembled proteinnanoparticle will include multiple copies of the heterologous carrier.

In further embodiments, the self-assembled protein nanoparticle isconjugated to a vaccine antigen.

V. POLYNUCLEOTIDES AND EXPRESSION

Polynucleotides encoding a disclosed fusion protein that forms aself-assembling protein nanoparticle carrier or self-assembling proteinnanoparticle are also provided. These polynucleotides include DNA, cDNAand RNA sequences which encode the fusion protein. The genetic code canbe used to construct a variety of functionally equivalent nucleic acids,such as nucleic acids which differ in sequence but which encode the sameprotein sequence, or encode a conjugate or fusion protein including thenucleic acid sequence.

In several embodiments, the nucleic acid molecule encodes a precursor ofa disclosed fusion protein and/or nanoparticle subunit, that, whenexpressed in cells under appropriate conditions, is processed andself-assembles into the protein nanoparticle carrier or proteinnanoparticle. For example, the nucleic acid molecule can encode aN-terminal signal sequence for entry into the cellular secretory systemthat is proteolytically cleaved in the during processing of the fusionprotein.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g., Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013).

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

The polynucleotides encoding a disclosed fusion protein and/ornanoparticle subunit can include a recombinant DNA which is incorporatedinto a vector into an autonomously replicating plasmid or virus or intothe genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (such as a cDNA) independent of other sequences. Thenucleotides can be ribonucleotides, deoxyribonucleotides, or modifiedforms of either nucleotide. The term includes single and double forms ofDNA.

Polynucleotide sequences encoding a disclosed fusion protein and/ornanoparticle subunit can be operatively linked to expression controlsequences. An expression control sequence operatively linked to a codingsequence is ligated such that expression of the coding sequence isachieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto, appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalsfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

DNA sequences encoding the disclosed fusion protein and/or nanoparticlesubunit can be expressed in vitro by DNA transfer into a suitable hostcell. The cell may be prokaryotic or eukaryotic. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. Methods of stable transfer, meaning thatthe foreign DNA is continuously maintained in the host, are known in theart.

Hosts can include microbial, yeast, insect and mammalian organisms.Methods of expressing DNA sequences having eukaryotic or viral sequencesin prokaryotes are well known in the art. Non-limiting examples ofsuitable host cells include bacteria, archea, insect, fungi (forexample, yeast), plant, and animal cells (for example, mammalian cells,such as human). Exemplary cells of use include Escherichia coli,Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9cells, C129 cells, 293 cells, Neurospora, and immortalized mammalianmyeloid and lymphoid cell lines. Techniques for the propagation ofmammalian cells in culture are well-known (see, e.g., Helgason andMiller (Eds.), 2012, Basic Cell Culture Protocols (Methods in MolecularBiology), 4^(th) Ed., Humana Press). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, andCOS cell lines, although cell lines may be used, such as cells designedto provide higher expression, desirable glycosylation patterns, or otherfeatures. In some embodiments, the host cells include HEK293 cells orderivatives thereof, such as GnTI^(−/−) cells (ATCC® No. CRL-3022), orHEK-293F cells.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques. In some embodiments, if the host isprokaryotic, such as, but not limited to, E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ method.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or viral vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding a disclosedantigen, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, ViralExpression Vectors, Springer press, Muzyczka ed., 2011). Appropriateexpression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines can be utilized.

Modifications can be made to a nucleic acid encoding a disclosed fusionprotein and/or nanoparticle subunit without diminishing its biologicalactivity. Some modifications can be made to facilitate the cloning orexpression of the fusion protein. Non-limiting examples of suchmodifications include termination codons, a methionine added at theamino terminus to provide an initiation site, additional amino acidsplaced on either terminus to create conveniently located restrictionsites, or additional amino acids (such as poly His) to aid inpurification steps.

VI. IMMUNOGENIC COMPOSITIONS

Immunogenic compositions comprising a disclosed immunogenic conjugateand a pharmaceutically acceptable carrier are also provided. Suchpharmaceutical compositions can be administered to subjects by a varietyof administration modes, for example, intramuscular, subcutaneous,intravenous, intra-arterial, intra-articular, intraperitoneal, orparenteral routes. IActual methods for preparing administrablecompositions are described in more detail in such publications asRemingtons Pharmaceutical Sciences, 19^(th) Ed., Mack PublishingCompany, Easton, Pa., 1995.

Thus, an immunogenic conjugate described herein can be formulated withpharmaceutically acceptable carriers to help retain biological activitywhile also promoting increased stability during storage within anacceptable temperature range. Potential carriers include, but are notlimited to, physiologically balanced culture medium, phosphate buffersaline solution, water, emulsions (e.g., oil/water or water/oilemulsions), various types of wetting agents, cryoprotective additives orstabilizers such as proteins, peptides or hydrolysates (e.g., albumin,gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g.,sodium glutamate), or other protective agents. The resulting aqueoussolutions may be packaged for use as is or lyophilized Lyophilizedpreparations are combined with a sterile solution prior toadministration for either single or multiple dosing.

Formulated compositions, especially liquid formulations, may contain abacteriostat to prevent or minimize degradation during storage,including but not limited to effective concentrations (usually ≤1% w/v)of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben,and/or propylparaben. A bacteriostat may be contraindicated for somepatients; therefore, a lyophilized formulation may be reconstituted in asolution either containing or not containing such a component.

The immunogenic compositions of the disclosure can contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate.

The immunogenic composition may optionally include an adjuvant toenhance an immune response of the host. Suitable adjuvants are, forexample, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-Aand derivatives or variants thereof, oil-emulsions, saponins, neutralliposomes, liposomes containing the vaccine and cytokines, non-ionicblock copolymers, and chemokines. Non-ionic block polymers containingpolyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POEblock copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) mayalso be used as an adjuvant (Newman et al., 1998, Critical Reviews inTherapeutic Drug Carrier Systems 15:89-142). These adjuvants have theadvantage in that they help to stimulate the immune system in anon-specific way, thus enhancing the immune response to a pharmaceuticalproduct.

In some embodiments, the immunogenic composition can be provided as asterile composition. The immunogenic composition typically contains aneffective amount of a disclosed immunogenic conjugate and can beprepared by conventional techniques. Typically, the amount ofimmunogenic conjugate in each dose of the immunogenic composition isselected as an amount which elicits or primes an immune response withoutsignificant, adverse side effects. In some embodiments, the immunogeniccomposition can be provided in unit dosage form for use to elicit orprime an immune response in a subject, for example, to prevent HIV-1infection in the subject. A unit dosage form contains a suitable singlepreselected dosage for administration to a subject, or suitable markedor measured multiples of two or more preselected unit dosages, and/or ametering mechanism for administering the unit dose or multiples thereof.

VII. METHODS OF INDUCING AN IMMUNE RESPONSE

The disclosed immunogenic conjugates and compositions including same canbe administered to a subject to induce an immune response to HIV-1 toprevent, inhibit, and/or treat an HIV-1 infection. The immune responsecan be a protective immune response, for example a response thatprevents or reduces subsequent infection with HIV-1. Elicitation of theimmune response can also be used to treat or inhibit infection andillnesses associated with HIV-1 infection. Thus, the disclosedimmunogenic conjugates and compositions including same can be used inmethods of preventing, inhibiting, or treating an HIV-1 infection. Inseveral embodiments, an effective amount of an immunogenic conjugate orcomposition including same can be administered to a subject in order togenerate a neutralizing immune response to HIV-1.

When inhibiting, treating, or preventing HIV-1 infection, the methodscan be used either to avoid infection in an HIV-1 seronegative subject(e.g., by inducing an immune response that protects against HIV-1infection), or to treat existing infection in an HIV-1 seropositivesubject. The HIV-1 seropositive subject may or may not carry a diagnosisof AIDS. Hence in some embodiments the methods involve selecting asubject at risk for contracting HIV-1 infection, or a subject at risk ofdeveloping AIDS (such as a subject with HIV-1 infection), andadministering a disclosed immunogenic conjugate or composition includingsame to the subject to elicit an immune response to HIV-1 in thesubject.

Treatment of HIV-1 by inhibiting HIV-1 replication or infection caninclude delaying the development of AIDS in a subject. Treatment ofHIV-1 can also include reducing signs or symptoms associated with thepresence of HIV-1 (for example, by reducing or inhibiting HIV-1replication). In some examples, treatment using the methods disclosedherein prolongs the time of survival of the subject.

Typical subjects intended for treatment with the therapeutics andmethods of the present disclosure include humans, as well as non-humanprimates and other animals. To identify subjects for prophylaxis ortreatment according to the methods of the disclosure, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease or condition, or to determine the statusof an existing disease or condition in a subject. These screeningmethods include, for example, conventional work-ups to determineenvironmental, familial, occupational, and other such risk factors thatmay be associated with the targeted or suspected disease or condition,as well as diagnostic methods, such as various ELISA and otherimmunoassay methods to detect and/or characterize HIV-1 infection. Theseand other routine methods allow the clinician to select patients in needof therapy using the methods and pharmaceutical compositions of thedisclosure.

The disclosed immunogenic conjugates and compositions including same canbe used in coordinate (or prime-boost) immunization protocols orcombinatorial formulations. In certain embodiments, novel combinatorialimmunogenic compositions and coordinate immunization protocols employseparate immunogenic conjugate or formulations, each directed towardeliciting an anti-HIV-1 immune response, such as an immune response toHIV-1 Env protein. Separate immunogenic conjugates and compositionsincluding same that elicit the anti-HIV-1 immune response can becombined in a polyvalent immunogenic composition administered to asubject in a single immunization step, or they can be administeredseparately (in monovalent immunogenic compositions) in a coordinateimmunization protocol.

In one embodiment, a suitable immunization regimen includes at least twoseparate inoculations with one or more immunogenic compositionsincluding a disclosed immunogen, with a second inoculation beingadministered more than about two, about three to eight, or about four,weeks following the first inoculation. A third inoculation can beadministered several months after the second inoculation, and inspecific embodiments, more than about five months after the firstinoculation, more than about six months to about two years after thefirst inoculation, or about eight months to about one year after thefirst inoculation. Periodic inoculations beyond the third are alsodesirable to enhance the subject's “immune memory.” The adequacy of theimmunization parameters chosen, e.g., formulation, dose, regimen and thelike, can be determined by taking aliquots of serum from the subject andassaying antibody titers during the course of the immunization program.Alternatively, the T cell populations can be monitored by conventionalmethods. In addition, the clinical condition of the subject can bemonitored for the desired effect, e.g., prevention of HIV-1 infection orprogression to AIDS, improvement in disease state (e.g., reduction inviral load), or reduction in transmission frequency to an uninfectedpartner. If such monitoring indicates that immunization is sub-optimal,the subject can be boosted with an additional dose of immunogeniccomposition, and the immunization parameters can be modified in afashion expected to potentiate the immune response.

It is contemplated that there can be several boosts, and that each boostcan be a different HIV-1 immunogen. It is also contemplated in someexamples that the boost may be the same immunogen as another boost, orthe prime.

In some embodiments, the prime comprises administration of animmunogenic conjugate as described herein, and the boost (or boosts)comprises administration a recombinant HIV-1 Env ectodomain trimer thatis stabilized in a prefusion mature closed conformation, for example, asdescribed in PCT App. No. PCT/US2015/048729 (incorporated by referenceherein in its entirety).

The prime and the boost can be administered as a single dose or multipledoses, for example, two doses, three doses, four doses, five doses, sixdoses or more can be administered to a subject over days, weeks ormonths. Multiple boosts can also be given, such one to five, or more.Different dosages can be used in a series of sequential inoculations.For example, a relatively large dose in a primary inoculation and then aboost with relatively smaller doses. The immune response against theselected antigenic surface can be generated by one or more inoculationsof a subject.

In several embodiments, the immunogenic conjugate can be administered tothe subject simultaneously with the administration of an adjuvant. Inother embodiments, the immunogenic conjugate can be administered to thesubject after the administration of an adjuvant and within a sufficientamount of time to elicit the immune response.

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject, orthat elicit a desired response in the subject (such as a neutralizingimmune response). Suitable models in this regard include, for example,murine, rat, porcine, feline, ferret, non-human primate. Alternatively,effective dosages can be determined using in vitro models (for example,immunologic and histopathologic assays). Using such models, ordinarycalculations and adjustments can be used to determine an appropriateconcentration and dose to administer an effective amount of thecomposition (for example, amounts that are effective to elicit a desiredimmune response or alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the immunogenic conjugate may simply inhibit or enhance one ormore selected biological activities correlated with a disease orcondition, as set forth herein, for either therapeutic or diagnosticpurposes.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site. Higher or lower concentrations can beselected based on the mode of delivery, for example, trans-epidermal,rectal, oral, pulmonary, or intranasal delivery versus intravenous orsubcutaneous delivery. The actual dosage of disclosed immunogenicconjugate will vary according to factors such as the disease indicationand particular status of the subject (for example, the subject's age,size, fitness, extent of symptoms, susceptibility factors, and thelike), time and route of administration, other drugs or treatments beingadministered concurrently, as well as the specific pharmacology of thecomposition for eliciting the desired activity or biological response inthe subject. Dosage regimens can be adjusted to provide an optimumprophylactic or therapeutic response.

A non-limiting range for an effective amount of the disclosedimmunogenic conjugate within the methods and immunogenic compositions ofthe disclosure is about 0.0001 mg/kg body weight to about 10 mg/kg bodyweight, such as about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg,about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg,about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg,about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg,about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg,about 5 mg/kg, or about 10 mg/kg, for example, 0.01 mg/kg to about 1mg/kg body weight, about 0.05 mg/kg to about 5 mg/kg body weight, about0.2 mg/kg to about 2 mg/kg body weight, or about 1.0 mg/kg to about 10mg/kg body weight. In some embodiments, the dosage includes a set amountof a disclosed immunogenic conjugate such as from about 1-300 μg, forexample, a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 μg.

The dosage and number of doses will depend on the setting, for example,in an adult or anyone primed by prior HIV-1 infection or immunization, asingle dose may be a sufficient booster. In naïve subjects, in someexamples, at least two doses would be given, for example, at least threedoses. In some embodiments, an annual boost is given, for example, alongwith an annual influenza vaccination.

For any application, immunization with a disclosed immunogenic conjugatecan be combined with anti-retroviral therapy, such as HAART.Antiretroviral drugs are broadly classified by the phase of theretrovirus life-cycle that the drug inhibits. The therapeutic agents canbe administered before, during, concurrent to and/or after retroviraltherapy. In some embodiments, the therapeutic agents are administeredfollowing a course of retroviral therapy. The disclosed therapeuticagents can be administered in conjunction with nucleoside and nucleotidereverse transcriptase inhibitors (nRTI), non-nucleoside reversetranscriptase inhibitors (NNRTI), protease inhibitors, Entry inhibitors(or fusion inhibitors), Maturation inhibitors, or a broad spectruminhibitors, such as natural antivirals. Exemplary agents includelopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabineand efavirenz.

HIV-1 infection does not need to be completely eliminated or reduced orprevented for the methods to be effective. For example, elicitation ofan immune response to HIV-1 with one or more of the disclosedimmunogenic conjugates (or an immunization protocol involving adisclosed immunogenic conjugate) can reduce or inhibit HIV-1 infectionby a desired amount, for example, by at least 10%, at least 20%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100% (elimination orprevention of detectable HIV-1 infected cells), as compared to HIV-1infection in the absence of the therapeutic agent. In additionalexamples, HIV-1 replication can be reduced or inhibited by the disclosedmethods. HIV-1 replication does not need to be completely eliminated forthe method to be effective. For example, the immune response elicitedusing one or more of the disclosed immunogens can reduce HIV-1replication by a desired amount, for example, by at least 10%, at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100% (elimination orprevention of detectable HIV-1 replication), as compared to HIV-1replication in the absence of the immune response.

To successfully reproduce itself, HIV-1 must convert its RNA genome toDNA, which is then imported into the host cell's nucleus and insertedinto the host genome through the action of HIV-1 integrase. BecauseHIV-1's primary cellular target, CD4+ T-Cells, can function as thememory cells of the immune system, integrated HIV-1 can remain dormantfor the duration of these cells' lifetime. Memory T-Cells may survivefor many years and possibly for decades. This latent HIV-1 reservoir canbe measured by co-culturing CD4+ T-cells from infected patients withCD4+ T-Cells from uninfected donors and measuring HIV-1 protein or RNA(See, e.g., Archin et al., AIDS, 22:1131-1135, 2008). In someembodiments, the provided methods induce an immune response in thesubject that reduces or eliminates of the latent reservoir of HIV-1infected cells in a subject. For example, a reduction of at least 10%,at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or even at least 100%(elimination of detectable HIV-1) of the latent reservoir of HIV-1infected cells in a subject, as compared to the latent reservoir ofHIV-1 infected cells in a subject in the absence of immunization withone or more of the provided immunogenic conjugates.

Following immunization of a subject, serum can be collected from thesubject at appropriate time points, frozen, and stored forneutralization testing. Methods to assay for neutralization activity andinclude, but are not limited to, plaque reduction neutralization (PRNT)assays, microneutralization assays, flow cytometry based assays,single-cycle infection assays (e.g., as described in Martin et al.(2003) Nature Biotechnology 21:71-76), and pseudovirus neutralizationassays (e.g., as described in Georgiev et al. (Science, 340, 751-756,2013), Seaman et al. (J. Virol., 84, 1439-1452, 2005), and Mascola etal. (J. Virol., 79, 10103-10107, 2005), each of which is incorporated byreference herein in its entirety. In some embodiments, the serumneutralization activity can be assayed using a panel of HIV-1pseudoviruses as described in Georgiev et al., Science, 340, 751-756,2013 or Seaman et al. J. Virol., 84, 1439-1452, 2005. Briefly,pseudovirus stocks are prepared by co-transfection of 293T cells with anHIV-1 Env-deficient backbone and an expression plasmid encoding the Envgene of interest. The serum to be assayed is diluted in Dulbecco'smodified Eagle medium-10% FCS (Gibco) and mixed with pseudovirus. After30 min, 10,000 TZM-bl cells are added, and the plates are incubated for48 hours. Assays are developed with a luciferase assay system (Promega,Madison, Wis.), and the relative light units (RLU) are read on aluminometer (Perkin-Elmer, Waltham, Mass.). To account for background, acutoff of ID₅₀≥40 can be used as a criterion for the presence of serumneutralization activity against a given pseudovirus.

In some embodiments, administration of an effective amount of one ormore of the disclosed the immunogenic conjugates to a subject elicits aneutralizing immune response in the subject, wherein serum from thesubject neutralizes, with an ID₅₀≥40, at least 10% (such as at least15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least70%) of pseudoviruses is a panel of pseudoviruses including the HIV-1Env proteins listed in Table S5 or Table S6 of Georgiev et al. (Science,340, 751-756, 2013), or Table 1 of Seaman et al. (J. Virol., 84,1439-1452, 2005).

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Immunogenic Conjugate of HIV-1 Env Fusion Peptides Conjugatedto a Self-Assembling Protein Nanoparticle Carrier

This example illustrates immunogenic conjugates including HIV-1 Envfusion peptides conjugated to a self-assembling protein nanoparticlecarrier. The immunogenic conjugate provides a multivalent platform withsuperior binding capability for engaging HIV-1 Env fusionpeptide-directed broadly neutralizing antibodies and can be used, forexample, to prime an immune response in a subject that targets the HIV-1Env fusion peptide epitope.

FIG. 1 illustrates the design of certain embodiments of the immunogenicconjugate. As shown in FIG. 1A, the self-assembling protein nanoparticlecarrier is a multimer of fusion proteins, each including aself-assembling protein nanoparticle subunit fused to a heterologouscarrier protein. In some embodiments, the fusion protein can furtherinclude a T-cell helper epitope (FIG. 1B), which is then included in theself-assembling protein nanoparticle carrier. The location of theT-cell-helper epitope can be varied in the fusion protein. As shown inFIGS. 1C-1I, the HIV-1 Env fusion peptides (FP) are conjugated to theself-assembling protein nanoparticle carrier. FIGS. 1G-1I illustrateadditional embodiments that further include a targeting moiety thattargets the immune system in a subject to enhance the immune response tothe HIV-1 Env fusion peptide on the immunogenic conjugate. The HIV-1 Envfusion peptides and the targeting moiety can be conjugated to anysuitable aspect of the self-assembling protein nanoparticle carrier. Insome instances, sulfosuccinimidyl (4-iodoacetyl)aminobenzoate(Sulfo-SIAB) conjugation chemistry is used to conjugate the HIV-1 Envfusion peptides and/or the targeting moiety to exposed lysine residuesof the self-assembling protein nanoparticle carrier.

Example 2 HIV-1 Env Fusion Peptide Immunization Using a NanoparticleFormat

To illustrate the effectiveness of the nanoparticle format forimmunization with the HIV-1 Env fusion peptide, the FP8 peptide(AVGIGAVF, residues 1-8 of SEQ ID NO: 1) was conjugated to KLHnanoparticles or to KLH monomeric subunits (FIG. 2). The resultingconjugates were administrated to mice, and immune sera assessed forbinding to BG505 HIV-1 Env trimer. As shown in FIG. 2 the nanoparticlebased immunogen elicited a much greater immune response to the HIV-1 Envtrimer than the subunit-based immunogen.

Example 3 Nanoparticle-Carriers for Display of Vaccine Antigens

This example illustrates self-assembling protein nanoparticles fused toheterologous carrier proteins for display of vaccine antigens.

Using structure based design, protein nanoparticle subunits wereselected for genetic fusion with heterologous carrier proteins by avariety of peptide linkers.

The nanoparticle subunits were ferritin subunits, lumazine synthasesubunits, encapsulin subunits, DNA starvation/stationary phaseprotection protein subunits, T4 fibritin subunits, Sulfur OxygenaseReductase subunits, Bacteriophage Q Beta Capsid protein (qbeta)subunits, Dihydrolipoyl transacetylase protein (e2p) subunits,Phosphopantetheine Adenylyltransferase (6ccq) subunits, GlutamateSynthase (1f52) subunits, Calcium/calmodulin dependent protein kinaseIIa (CaMKIIa) C-terminal fragment (5U6Y) subunits, HIV capsidoligomerization domain subunits, Hexamer subunits, Acinetobacter phageAP205 subunits, and Hepatitis B capsid subunits.

The heterologous carrier proteins were tetanus toxin heavy chain Cfragment (rTT), diphtheria toxin variant CRM197 (CRM197), H. influenzaeprotein D, Keyhole Limpet Hemocyanin (KLH) functional unit,Meningococcal outer membrane protein complex protein, Outer-membranelipoprotein carrier protein, and Cholera toxin B subunit.

The linkers were an IgG hinge, a camel IgG2a hinge, a CD8 hinge, and aglycine serine linker

Combinations of self-assembling nanoparticle subunit, heterologouscarrier, and linker were assessed computationally for formation of amultimerized protein nanoparticle with the heterologous carrier proteinfused to each subunit displayed on the exterior surface of thenanoparticle. These design assays led to the identification of thefusion proteins set forth as SEQ ID NOs: 72-219, 246-257, and 331-397,which self-assemble to form nanoparticle carrier proteins.

To illustrate the nanoparticle-forming capacity of the identified fusionproteins, a fusion protein containing a lumazine synthase nanoparticlesubunit fused to rTT by a 20 amino acid peptide linker (LS-20-rTT) wasassessed for nanoparticle self-assembly (FIG. 3). The fusion protein isdepicted in FIG. 3A and the sequence is set forth as SEQ ID NO: 73. Amammalian expression construct encoding LS-20-rTT was expressed inmammalian cells using a standard protocol for generating lumazinesynthase nanoparticles (see, Zhang et al. “X-ray structure analysis andcrystallographic refinement of lumazine synthase from thehyperthermophile Aquifex aeolicus at 1.6 Å resolution: determinants ofthermostability revealed from structural comparisons.” J Mol Biol.,306(5):1099-114, 2001 and Duan et al., “Glycan Masking Focuses ImmuneResponses to the HIV-1 CD4-Binding Site and Enhances Elicitation ofVRC01-Class Precursor Antibodies,” Immunity, 49(2):301-311, 2018, eachof which is incorporated by reference herein), and the resultingnanoparticles self-assemble in the tissue culture media. Thenanoparticles were purified and separated by size-exclusionchromatography (FIG. 3B) and assessed by electron microscopy (FIG. 3C).As shown, the resulting nanoparticles are uniform and stable, and readyfor conjugation with vaccine antigen.

Additionally, a fusion protein containing a lumazine synthasenanoparticle subunit fused to rTT by an IgG hinge linker (LS-hinge2-rTT)was assessed for nanoparticle self assembly (FIG. 4). The fusion proteinsequence is set forth as SEQ ID NO: 362. A mammalian expressionconstruct encoding LS-hinge2-rTT was expressed in mammalian cells asabove, and the resulting nanoparticles were purified and assessed byelectron microscopy. Again, the resulting nanoparticles are uniform andstable, and ready for conjugation with vaccine antigen.

Additionally, a fusion protein containing a phosphopantetheineadenylyltransferase nanoparticle subunit was assessed for nanoparticleself assembly (FIG. 5). This fusion protein contained two carrierproteins: the fusion protein contained H. influenzae protein D carrierfused to the phosphopantetheine adenylyltransferase nanoparticle subunitfused to rTT carrier (HiD-6CCQ-rTT). The fusion protein sequence is setforth as SEQ ID NO: 179. A mammalian expression construct encodingHiD-6CCQ-rTT was expressed in mammalian cells as above, and theresulting nanoparticles were purified and assessed by electronmicroscopy. The observed particles were generally consistent in size andshape with the known phosphopantetheine adenylyltransferase crystalstructure (PDB 6CCQ). Again, the resulting nanoparticles are uniform andstable, and ready for conjugation with vaccine antigen.

Example 4 Conjugation of HIV-1 Env Fusion Peptide to NanoparticleCarrier

The following provides a non-limiting example of a method of conjugatinga HIV-1 Env fusion peptide (FP8, AVGIGAVF, residues 1-8 of SEQ ID NO: 1)to a self-assembling protein nanoparticle carrier (formed fromLS-PADRE-Env31-rTT fusion proteins, SEQ ID NO: 76) via asulfosuccinimidyl (4-iodoacetyl)aminobenzoate (Sulfo-SIAB) linker. Theprotocol used to link the fusion peptide to carrier can be performedaccording to standard methods (see, e.g., Hermanson. BioconjugationTechniques, 3^(rd) ed., Chap. 6, p. 306-308. Academic Press, 2013).Briefly, the conjugation protocol includes:

Expression of the Self-Assembling Protein Nanoparticle Carrier

An expression construct encoding the LS-PADRE-Env31-rTT fusion protein(SEQ ID NO: 76) including an N-terminal signal peptide is expressed inHEK 293 Freestyle cells. The fusion proteins are secreted from the cellsand self-assemble into the protein nanoparticle carrier in thesupernatant. The resulting protein nanoparticle carrier is purifiedusing chromatography procedures, including anion exchange followed bysize exclusion chromatography.

Activation of LS-PADRE-Env31-rTT Nanoparticle Carrier:

-   -   1. Prepare 10 mM stock of sulfo-SIAB crosslinker    -   2. Prepare a 1 mg/mL LS-PADRE-Env31-rTT nanoparticle carrier        stock in conjugation buffer (10% glycerol, 50 mM Na/KPO₄ buffer,        pH 8.5, 1 mM EDTA).    -   3. Add sulfo-SIAB to LS-PADRE-Env31-rTT nanoparticle carrier        using a 1:1 molar ratio of crosslinker to total Lys on        LS-PADRE-Env31-rTT nanoparticle carrier    -   4. Let reaction proceed at 25° C. (room temperature) for 1 hr.    -   5. At 4° C., pass through a 10 ml Zebra Spin Desalting Column,        7K MWCO (Thermofisher) to remove low molecular weight compounds.

Conjugation of Peptide to Activated Carrier:

-   -   1. Prepare a 12 mM stock of FP8 peptide.    -   2. Allow activated LS-PADRE-Env31-rTT nanoparticle carrier to        warm up to 25° C. (room temperature). Gradually add peptide to        activated carrier using a 1:1 (w/w) ratio.    -   3. Spin for 2 min; use supernatant and discard precipitate.    -   4. Incubate reaction supernatant at 4° C. overnight.    -   5. Use a 10 ml Zebra Spin Desalting Column, 7K MWCO        (Thermofisher) to remove low molecular weight compounds.    -   6. Dialyze conjugate against 1×PBS.    -   7. Analyze product: degree of conjugation by mass spectrometry        and antigenic properties by Octet.

Following purification of the FPB-LS-PADRE-Env31-rTT nanoparticlecarrier conjugate, antigenicity can be assessed by binding to fusionpeptide specific antibody VRC34.

The conjugation protocol and chemistry illustrated in this example canreadily be extended to other fusion peptide sequences and other carrierproteins.

Example 5 Nanoparticle-Carriers Conjugated to Vaccine Antigens andRelated Immunization Assays

This example illustrates self-assembling protein nanoparticles fused toa heterologous carrier and conjugated to vaccine antigens (HIV-1 Envfusion peptide) and immunization therewith.

For the assays described in this example, the nanoparticle subunit waslinked to the heterologous carrier by isopeptide bond using thespytag/spycatcher linkage system. FIG. 6 depicts the construction andpurification protocol. rTT carrier was genetically fused to thespycatcher tag, and the lumazine synthase subunit was genetically fusedto the spytag. The sequence of the LS-spytag fusion is provided as SEQID NO: 399. The sequence of the rTT-spycatcher fusion is provided as SEQID NO: 407. The rTT-spyC fusion protein was produced and purified, andlumazine synthase nanoparticles formed form the LS-spytag fusion wereproduced purified. The rTT-spyC fusion protein and the lumazine synthasenanoparticles formed form the LS-spytag fusion were mixed, allowing thespytag/spycatcher proteins to spontaneously join by isopeptide bond,resulting in a lumazine synthase nanoparticle linked to rTT via thespycatcher/tag linker Subsequently, the FP8 fusion peptide wasconjugated to the purified nanoparticle-carrier by a PEG linker

The structure of rTT-spyC, LS-SpyT, LS-Spy-rTT, and LS-Spy-rTT-FP8 wereassessed by EM (FIG. 7). Further, the LS-Spy-rTT-FP8 nanoparticlecarrier was assessed for the number of conjugated HIV-1 Env fusionpeptides using ITC, and this was compared to the corresponding number ofHIV-1 Env fusion peptides conjugated to monomeric rTT (FIG. 8). Theresults show that each FP-rTT monomer entity has six competent VRC34.01Fab binding sites, whereas each LS-Spy-rTT-FP8v1/PEG2 nanoparticlecarrier has 152-402 competent VRC34.01 Fab binding sites. The VRC34.01antibody specifically binds to the HIV-1 Env fusion peptide.

The immunogenicity of the LS-Spy-rTT-FP8v1/PEG2 nanoparticle carrier wasassessed in a mouse model. The immunization protocol is shown in FIG. 9.For the first three immunizations (weeks 0, 3, and 6), mice received a25 μg dose of either FP8v1-rTT monomer (Groups 1 and 2) orLS-Spy-rTT-FP8v1/PEG2 nanoparticle carrier (Groups 3 and 4). For thefollowing three immunizations, mice received a 25 μg dose of eitherBG505 DS-SOSIP trimer (Groups 1 and 3) or the BG505 DS-SOSIP trimerconjugated to a lumazine synthase nanoparticle (Groups 2 and 4). BG505DS-SOSIP trimer is a known HIV-1 Env immunogen described in Kwon et al.(“Crystal structure, conformational fixation and entry relatedinteractions of mature ligand-free HIV-1 Env,” Nat Struct Biol.,22(7):522-531, 2015, incorporated by reference herein). For these assaysBG505 DS-SOSIP trimer was linked to lumazine synthase nanoparticles bystandard conjugation chemistry. Blood was drawn at weeks 0, 2, 5, 8, 11,14, and 17.

Thus, this immunization assay interrogates the ability of theLS-Spy-rTT-FP8v1/PEG2 nanoparticle carrier to generate an immuneresponse in an animal model, and also whether this construct can primean immune response for subsequent immunization with HIV-1 Env trimer.

As shown in FIG. 10, the LS-Spy-rTT-FP8v1/PEG2 immunogen elicited a farsuperior immune response to HIV-1 Env fusion peptide compared tomonomeric FP-rTT (FIGS. 10A and 10B), and also provided superior primingfor subsequent immunization with the BG505 trimer or BG505 trimer onlumazine synthase particle. These results illustrate the effectivenessof the self-assembled protein nanoparticle carrier fusion for use as aimmunization tool.

Example 6 Disulfide-Stabilized Nanoparticle Subunits for NanoparticleCarriers

This example illustrates self-assembling protein nanoparticles fused toheterologous carrier proteins for display of vaccine antigens that aremodified to contain a non-native disulfide bond to increase retention ofthe nanoparticle format.

Using structure based design, self-assembling protein nanoparticlesubunits were mutated to contain one or more cysteine substitutions tointroduce a non-native disulfide bond that stabilizes the correspondingnanoparticle formed by the subunits. Stabilization increases resistanceto disassembly of the nanoparticle compared to a corresponding nativesubunit sequence under similar conditions. The mutations were assessedcomputationally to determine whether they would form a disulfide bondthat would stabilize the resulting nanoparticle.

Based on this assessment, ferritin subunits set forth as SEQ ID NOs:258-305, lumazine synthase subunits set forth as SEQ ID NOs: 306-312,encapsulin subunits set forth as SEQ ID NOs: 313-315, Acinetobacterphage AP205 subunits set forth as SEQ ID NOs: 317-320, and Hepatitis Bcapsid subunits set forth as SEQ ID NOs: 322-326, were identified, whichself-assemble to form nanoparticles containing one or more non-nativedisulfide bonds that stabilize that nanoparticle relative tonanoparticles formed from unmodified subunits. Specific examples of thedisulfide stabilized protein nanoparticles fused to carrier proteins areprovided as SEQ ID NOs: 331-354, 369-387, 394-397.

To illustrate the nanoparticle-forming capacity of subunits containingthe indicated disulfide bonds, an encapsulin subunit containingG53C-R94C mutations to introduce a stabilizing disulfide bond was fusedto a spytag, expressed in cells and the corresponding self-assemblednanoparticles were purified and mixed with rTT-spycatcher (FIGS. 11-13)to form encapsulin-rTT nanoparticle carriers, with the carrier proteinlinked to the nanoparticle via the spytag/catcher isopeptide bond. Thesequence of the encapsulin G53C-R94C spytag fusion is provided as SEQ IDNO: 410, and the sequence of the rTT-spycatcher is provided as SEQ IDNO: 407. The purified nanoparticle-carrier was conjugated to FP8 fusionprotein using a SIAB linker FIG. 13 shows by EM that the resulting HIV-1Env fusion peptide nanoparticle carrier is uniform and stable.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described embodiments. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An immunogenic conjugate, comprising: a self-assemblingprotein-nanoparticle carrier comprising a multimer of fusion proteins,wherein each fusion protein comprises a self-assembling proteinnanoparticle subunit fused to a heterologous carrier protein, andwherein the fusion proteins self-assemble to form the self-assemblingprotein-nanoparticle carrier; and HIV-1 Env fusion peptides conjugatedto the self-assembling protein-nanoparticle carrier, wherein the HIV-1Env fusion peptides comprise, from the N-terminus, the amino acidsequence of residue 512 to one of residues 514-521 of a humanimmunodeficiency virus type 1 (HIV-1) Envelope (Env) protein accordingto the HXB2 numbering system; and wherein the immunogen elicits animmune response to HIV-1 Env. 2.-31. (canceled)
 32. A recombinantself-assembling nanoparticle subunit, comprising: a lumazine synthasenanoparticle subunit comprising cysteine substitutions to introduce oneor more non-native disulfide bonds to increase stability of thenanoparticle, wherein the cysteine substitutions comprise 121C and 131Csubstitutions, 121CG and 131C substitutions, 121GC and 131Csubstitutions, 7C and 40C substitutions, 3C and 50C substitutions, 82Cand 131CG substitutions, 5C and 52C substitutions, or 95C and A101Csubstitutions, or a combination thereof, wherein residue numberingcorresponds to a reference lumazine synthase subunit set forth as SEQ IDNO: 25; an encapsulin nanoparticle subunit comprising cysteinesubstitutions to introduce one or more non-native disulfide bonds toincrease stability of the nanoparticle, wherein the cysteinesubstitutions comprise 53C and 94C substitutions, 53C and 96Csubstitutions, or 146C and 185C substitutions, or a combination thereof,wherein residue numbering corresponds to a reference lumazine synthasesubunit set forth as SEQ ID NO: 43; an Acinetobacter phage AP205nanoparticle subunit comprising cysteine substitutions to introduce oneor more non-native disulfide bonds to increase stability of thenanoparticle, wherein the cysteine substitutions comprise a T81Csubstitution, 53C and 100C substitution, or 82C and 80C substitutions,or a combination thereof, wherein residue numbering corresponds to areference lumazine synthase subunit set forth as SEQ ID NO: 316; or aHepatitis B capsid protein nanoparticle subunit comprising cysteinesubstitutions to introduce one or more non-native disulfide bonds toincrease stability of the nanoparticle, wherein the cysteinesubstitutions comprise 25C and 127C substitutions, 14C and 36Csubstations, 29C and 127C substitutions, 18C and 36C substitutions, or29C and 127C substitutions, or a combination thereof, wherein residuenumbering corresponds to a reference lumazine synthase subunit set forthas SEQ ID NO:
 321. 33. A recombinant self-assembling nanoparticlesubunit, comprising: a ferritin nanoparticle subunit comprising orconsisting of the amino acid sequence set forth as any one of SEQ IDNOs: 258-305; a lumazine synthase nanoparticle subunit comprising orconsisting of the amino acid sequence set forth as any one of SEQ IDNOs: 306-312; an encapsulin nanoparticle subunit comprising orconsisting of the amino acid sequence set forth as any one of SEQ IDNOs: 313-315; a Acinetobacter phage AP205 protein subunit comprising orconsisting of the amino acid sequence set forth as any one of SEQ IDNOs: 317-320; or a Hepatitis B capsid protein subunit comprising orconsisting of the amino acid sequence set forth as any one of SEQ IDNOs: 322-326.
 34. The recombinant self-assembling nanoparticle subunitof claim 33, wherein the recombinant self-assembling nanoparticlesubunit is fused to a heterologous carrier protein.
 35. The recombinantself-assembling nanoparticle subunit of claim 34, wherein theheterologous carrier protein is selected from any one of a tetanus toxinheavy chain C fragment, a diphtheria toxin variant CRM197, and an H.influenzae protein D, a Keyhole Limpet Hemocyanin (KLH) functional unit,a Meningococcal outer membrane protein complex protein, anOuter-membrane lipoprotein carrier protein, or a Cholera toxin Bsubunit.
 36. The recombinant self-assembling nanoparticle subunit ofclaim 35, wherein the heterologous carrier protein is the tetanus toxinheavy chain C fragment.
 37. A nucleic acid molecule encoding therecombinant self-assembling nanoparticle subunit of claim
 32. 38. Arecombinant self-assembling nanoparticle comprising the recombinantself-assembling nanoparticle subunit of claim
 32. 39. The recombinantself-assembling nanoparticle of claim 38, conjugated to a vaccineantigen.
 40. An immunogenic composition comprising the recombinantself-assembling nanoparticle of claim
 39. 41. A method for generating animmune response to a vaccine antigen in a subject, comprisingadministering to the subject an effective amount of the immunogeniccomposition of claim 40 to generate the immune response.
 42. A nucleicacid molecule encoding the recombinant self-assembling nanoparticlesubunit of claim
 33. 43. A recombinant self-assembling nanoparticlecomprising the recombinant self-assembling nanoparticle subunit of claim33.
 44. The recombinant self-assembling nanoparticle of claim 33,conjugated to a vaccine antigen.
 45. An immunogenic compositioncomprising the recombinant self-assembling nanoparticle of claim
 44. 46.A method for generating an immune response to a vaccine antigen in asubject, comprising administering to the subject an effective amount ofthe immunogenic composition of claim 45 to generate the immune response.